Bivalent IgG Research Articles

Differential roles of CD36 and alphavbeta5 integrin in photoreceptor phagocytosis by the retinal pigment epithelium.

Retinal pigment epithelial (RPE) cells employ alphavbeta5 integrin and CD36 receptors to phagocytose photoreceptor outer segment fragments (OS). We explored special properties of RPE phagocytosis to identify the contribution of CD36 to RPE phagocytosis measuring effects of CD36 antibodies on OS binding and internalization kinetics. Early, CD36 antibodies had no effect on OS binding or internalization. Both control and CD36 antibody treated RPE initiated internalization approximately 2 hours after OS challenge. Later, bivalent CD36 IgG accelerated OS engulfment while monovalent Fab fragments inhibited engulfment. Cross-linking Fab fragments restored the accelerating activity of intact IgG. Strikingly, antibodies were effective even if added to OS already bound by RPE. alphavbeta5 blocking antibody reduced OS binding equally well in the presence of CD36 antibodies but CD36 antibodies accelerated internalization of remaining bound OS. Furthermore, CD36 ligation at either apical or basal RPE surface partially substituted for soluble factors that are required for internalization but not for binding of OS at the RPE apical surface. Our results demonstrate that CD36 ligation is necessary and sufficient to activate the OS internalization mechanism of RPE. They suggest that CD36 acts as a signaling molecule in postbinding steps of RPE phagocytosis independently of the OS binding receptor alphavbeta5 integrin.

Intracellular Stability of Anti-caspase-3 Intrabodies Determines Efficacy in Retargeting the Antigen

Although intracellular antibodies (intrabodies) are being explored as putative therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address this issue, we generated a panel of five intrabodies, directed against catalytically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv fragments must fulfill to act as efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied approximately 5-fold (50-250 nm). Despite their substantial sequence similarity, only two of the five intrabodies were able to significantly accumulate intracellularly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyzed by urea denaturation studies. We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal. Our results demonstrate that the intrinsic stability of the intrabody, rather than its affinity for the antigen, dictates its intracellular efficacy.

Effects of nerve growth factor on visual cortical plasticity require afferent electrical activity.

It is known that administration of nerve growth factor (NGF) prevents the ocular dominance shift induced by monocular deprivation in the rat. To determine whether electrical activity in the visual afferent pathway is required for NGF effects on ocular dominance, we infused NGF into the cortex of animals subjected to complete monocular blockade of retinal discharges. Rats at the peak of the critical period received intravitreal tetrodotoxin (TTX) injections to silence activity in one eye for a period of 6-7 days; NGF was concurrently delivered into the visual cortex by means of osmotic minipumps. At the end of the treatment period, the ocular dominance distribution of cortical neurons was assessed by single-cell recordings. The results demonstrate that while infusion of NGF is effective in preventing the ocular dominance shift in lid-sutured rats, virtually no rescue can be observed in TTX-injected animals. Identical results were obtained when a specific agonist of the NGF receptor TrkA, the bivalent anti-rat TrkA IgG (RTA), was infused into the cortex in place of NGF. We conclude that NGF signalling via the TrkA receptor must be coupled to afferent electrical activity to produce its effects on the eye preference of cortical neurons. This suggests a generalized mechanism in which high-affinity neurotrophin receptor activation and afferent discharge interact to modulate neuronal plasticity in the developing visual cortex.

TrkA activation in the rat visual cortex by antirat trkA IgG prevents the effect of monocular deprivation.

It has been recently shown that intraventricular injections of nerve growth factor (NGF) prevent the effects of monocular deprivation in the rat. We have tested the localization and the molecular nature of the NGF receptor(s) responsible for this effect by activating cortical trkA receptors in monocularly deprived rats by cortical infusion of a specific agonist of NGF on trkA, the bivalent antirat trkA IgG (RTA-IgG). TrkA protein was detected by immunoblot in the rat visual cortex during the critical period. Rats were monocularly deprived for 1 week (P21-28) and RTA-IgG or control rabbit IgG were delivered by osmotic minipumps. The effects of monocular deprivation on the ocular dominance of visual cortical neurons were assessed by extracellular single cell recordings. We found that the shift towards the ipsilateral, non-deprived eye was largely prevented by RTA-IgG. Infusion of RTA-IgG combined with antibody that blocks p75NTR (REX), slightly reduced RTA-IgG effectiveness in preventing monocular deprivation effects. These results suggest that NGF action in visual cortical plasticity is mediated by cortical TrkA receptors with p75NTR exerting a facilitatory role.

Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: Influence of radiation dose and dose rate on toxicity and anti-tumor efficacy.

Whereas bivalent fragments have been widely used for radio-immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of (131)I-labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose-limiting organ toxicity and anti-tumor efficacy. The maximum tolerated doses (MTDs) and dose-limiting organ toxicities of the (131)I-labeled anti-CEA antibody MN-14 [IgG, F(ab')2 and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D-lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 (131)I-labeled immunoconjugates [IgG, F(ab')2 and Fab], the red marrow was the only dose-limiting organ; MTDs were 260 microCi for IgG, 1,200 microCi for F(ab')2 and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab')2. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti-tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments.

Only Selected Light Chains Combine with a Given Heavy Chain to Confer Specificity for a Model Glycopeptide Antigen

The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, library-derived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.

Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents.

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab')2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti-neuraminidase NC10 scFv-streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcore biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti-ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.

Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10 7 M −1 and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

Homodimerization of Erythropoietin Receptor by a Bivalent Monoclonal Antibody Triggers Cell Proliferation and Differentiation of Erythroid Precursors

AbstractErythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Several lines of evidence indicate that the most likely mechanism of EPO receptor (EPO-R) activation by EPO is homodimerization of the receptor on the surface of erythrocyte precursors. Therefore, we argued that it should be possible to raise EPO-R monoclonal antibodies (MoAbs) that would activate the receptor by dimerization and thus mimic EPO action. We have identified such an agonist MoAb (MoAb34) directed against the extracellular EPO binding domain of the EPO-R. This bivalent IgG antibody triggers the proliferation of EPO-dependent cell lines and induces differentiation of erythroid precursors in vitro. In contrast, the monovalent Fab fragment, which cannot dimerize the receptor, is completely inactive. The mechanism of receptor activation by homodimerization implies that at high ligand concentrations the formation of 1:1 receptor/ligand complexes is favored over 2:1 complexes, thereby turning the ligand agonist into an antagonist. Thus, EPO and MoAb34 should self-antagonize at high concentrations in both cell proliferation and differentiation assays. Our data indeed demonstrate that EPO and MoAb34 antagonize ligand-dependent cell proliferation with IC50 values of approximately 20 and 2 μmol/L, respectively. Erythroid colony formation (BFUe) is inhibited at MoAb34 concentrations above 1 μmol/L. Furthermore, we analyzed the MoAb34:EPO-R interaction using a mathematic model describing antibody-mediated receptor dimerization. The data for proliferation and differentiation activity were consistent with the receptor dimer formation on the cell surface predicted by the model.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments.

Polyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the VH-diversity(D)-JH and V kappa-J kappa gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro C gamma l and C kappa human expression system. These mAbs are virtually identical in their VH and V kappa gene segment sequences. First, we expressed the VH-D-JH and V kappa-J kappa genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or kappa chain with the mAb13-derived kappa or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived kappa V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived kappa V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1)-FR3 and complementarity determining region 3 (CDR3) regions of the H and kappa chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived kappa chain FR1-FR3, but not that of the kappa chain CDR3.(ABSTRACT TRUNCATED AT 400 WORDS)

Bispecific monoclonal antibody regulation of FcγRIII-Directed tumor cytotoxicity by large granular lymphocytes

Bispecific monoclonal antibodies (BsMAbs) prepared by somatic cell fusion bind monovalently to their targets and yet are extremely potent enhancers of target cell lysis by relevant effector cells. The mechanisms underlying this efficiency are not known. To investigate this property, we studied the ability of selected antibodies to modulate potentiation of tumor lysis by a bispecific antibody (CL158) which targets FcγRIII-expressing cells, via the 3G8 epitope, to malignant cells expressing CA19-9 antigen. Antibodies directed against the 3G8 and B73.1 epitopes of Fcγ RIII efficiently inhibited BsMAb-mediated SW948 tumor cell lysis by interleukin-2 (IL-2)-activated lymphocytes (PBLs). Unexpectedly, Leu 19 antibody reversed antibody-dependent but not antibody-independent lysis of 51Cr-labeled SW948 cells by IL-2-activated PBLs in a concentration-dependent fashion. Leu 19 binds to CD56, a neural cell adhesion molecule (N-CAM) isoform expressed by large granular lymphocytes (LGLs). The effects of Leu 19 on bispecific antibody promotion of lysis were due to competition for binding to the 3G8 epitope of Fcγ RIII and led to inhibition of binding between LGLs and SW948 cells. Leu 19 did not inhibit antibody-dependent lysis by the monospecific, bivalent IgG 2a variant of CA19-9 antibody. These studies show that competition assays can be useful in dissecting the relevant mechanisms underlying BsMAb-promoted lysis. Steric constraints between effector cell trigger molecules (i.e., Fcγ RIII) and CAM such as N-CAM may regulate the function of these molecules. Understanding the roles of diverse CAM in this phenomenon will facilitate efforts to expand and use defined effector cell populations with maximal lytic potential and to identify potentially responsive tumor phenotypes.

61. Identity of differentiation inhibiting factor for mouse myeloid leukemia cells with Nm23/nucleoside diphosphate kinase

Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

Dissociation of IgG antibody-mediated clumps ofTrypanosoma brucei gambiense by complement

Aggregated trypanosome masses that had formed in the region of equivalence in the absence of complement were readily dissociated by complement. Complement-mediated dissociation of the clumped trypanosomes in the area of equivalence released approximately 50% of the previously bound surface antigens. These antigens were capable of rebinding to new IgG antibody. Experiments indicated that complement deposition altered the bivalent IgG antibody in the immune complex into a univalent form. The occurrence of such an event in the presence of complement provides a great advantage to the infected host in the killing of pathogens in vivo, as it enables a greater number of antibodies to attach to surface antigens and subsequently initiate complement activity.

Polyvalent interaction of antibodies with bacterial cells

We have studied the physical-chemical characteristics of the interaction of peroxidase-labelled rabbit antibodies with Bacillus sp. bacterial cells. The antibodies are able to bind bivalently with two antigen sites on the bacterial cells with the formation of intramolecular “cyclic” complexes. A kinetic model is proposed suggesting the existence of monovalent and bivalent cell surface antigens. The equilibrium constant of the bivalent IgG binding to the bacterial cell is by two orders of magnitude higher as compared to monovalent Fab fragments. The intramolecular reaction between the free active site of the monovalently bound antibody and a free antigen site on the cell surface is the rate limiting step of the polyvalent interaction. Formation of the cyclic complexes seems to be accompanied by essential tension of bonds and deformation of the IgG molecule. Agglutination of bacterial cells was also studied. The cell agglomerate size dependence on the antibody concn has a threshold. Agglutination proceeds under conditions where the antigen-antibody binding on the cell surface is far from equilibrium.

Cross-linking of Fc gamma receptors and surface antibodies. Theory and application.

B lymphocyte responses to the cross-linking of surface Ig (sIg) are known to be inhibited, when IgG is the cross-linking agent, by the concurrent binding of the Fc portion of the IgG to Fc gamma R. We present a mathematical framework for designing and analyzing experiments aimed at uncovering the inhibition mechanism(s). From our model, we calculate concentrations of receptors and ligands in the different cell surface states, at equilibrium or as a function of time. IgG can cross-link surface receptors in three ways, i.e., by bridging two sIg molecules without Fc binding, by bridging two sIg while binding as well to an Fc gamma R, and by binding to an Fc gamma R and only one sIg. We show how the concentrations or fractions of these distinct cross-linked states depend on experimentally manipulable variables, including the concentrations of intact IgG, bivalent and monovalent IgG fragments, and agents that block Fc binding. Then, using simple signal/response relationships, reflecting active and passive mechanisms of Fc-mediated inhibition, we simulate the results of a variety of experiments. In cases where published experimental results are available, we find that the qualitative predictions of our general model are consistent with the data and that comparisons of simulations with available data provide some quantitative information about the parameters governing the cell surface signaling events. In particular, comparison of model predictions with published experiments on the kinetics of IgG-induced inositol trisphosphate production indicate that sIg cross-links form more rapidly than sIg-Fc gamma R "co-cross-links." Further, IgG-sIg bonds stabilize Fc attachments, i.e., the dissociation of IgG from Fc gamma R is slowed significantly when the IgG is also cross-linked to sIg. Predictions of the model suggest other experiments and ways of presenting the data that will help to identify relationships between the molecular signaling events occurring on the cell surface and the various cellular responses.

Heparin binding domain of antithrombin III: characterization using a synthetic peptide directed polyclonal antibody

Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. We have previously proposed that the heparin binding site of ATIII resides within a region extending from amino acid residues 114-156 [Smith, J. W., & Knauer, D. J. (1987) J. Biol. Chem. 262, 11964-11972]. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation.

Transcriptional stimulation of the delta 1-crystallin gene by insulin-like growth factor I and insulin requires DNA cis elements in chicken.

Insulin-like growth factor I (IGF-I) and insulin regulate expression of the endogenous delta 1-crystallin gene in embryonic lens cells that express receptors for both peptides. To further analyze the transcriptional component of this hormonal effect, transient transfections of lens cells were prepared with DNA constructs containing deletions of the delta 1-crystallin promoter and the chloramphenicol acetyltransferase reporter gene. A 77-nucleotide DNA segment of the delta 1-crystallin promoter from nucleotide positions-120 to -43 confers sensitivity to insulin and IGF-I. The hormonal effect is dose-dependent, and maximal stimulation of promoter activity (2- to 2.5-fold induction) is obtained with 10(-8) M IGF-I and 10(-7) M insulin. Mobility-shift DNA-binding analysis shows specific binding of nuclear protein(s) to the delta 1-crystallin promoter DNA between positions -120 and +23, which appears to be regulated by IGF-I. An SP1-binding motif is involved in this DNA-protein interaction. The bivalent IgG fraction of an anti-insulin receptor antiserum (B-10), known to mimic insulin action in other systems, stimulates promoter activity to the same extent as insulin.

Immunochemical probing of the N-terminal segment on actin: the polymerization reaction

The N-terminal segment of actin contains a cluster of acidic residues which are implicated in macromolecular interactions of this protein. In this work, the interrelationship between the N-terminal segment and the polymerization of actin was studied by using affinity-purified antibodies directed against the first seven N-terminal residues on alpha-skeletal actin (S alpha N). The Fab fragments of these antibodies showed equal affinities for G- and F-actin while the bivalent IgG bound preferentially to the polymerized actin. As monitored by pyrene fluorescence measurements, the binding of Fab to G-actin did not alter the kinetics of the MgCl2-induced polymerization; IgG accelerated this reaction considerably. Consistent with these observations, the binding of Fab to F-actin did not change its morphological appearance in electron micrographs and had no effect on the stability and the rate of dissociation of actin filaments. These results are discussed in terms of their implications to the spatial relationship between the N-terminal segment and the rest of the molecule and the context of the polymerization reaction of actin in vitro and in vivo.

Application of fluorescence photobleaching recovery to assess complex formation between the two envelope proteins of Sendai virus in membranes of fused human erythrocytes

Fusion of human erythrocytes by Sendai virions is accompanied by lateral mobilization of the viral envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) in the target cell membrane; the dynamic parameters characterizing the lateral diffusion of F and HN in the fused cell membrane are identical [Henis, Y. I., & Gutman, O. (1987) Biochemistry 26, 812-819; Aroeti, B., & Henis, Y. I. (1988) Biochemistry 27, 5654-5661]. This identity raised the possibility that F and HN diffuse together in the cell membrane in mutual heterocomplexes. In order to investigate the possible formation of F-HN complexes in the target cell membrane, which could be important for the fusion process mediated by the viral envelope proteins, we combined fluorescence photobleaching recovery (FPR) measurements of the lateral mobility of the viral glycoproteins with antibody-mediated cross-linking of F or HN. After fusion, one viral glycoprotein type was immobilized by cross-linking with highly specific bivalent polyclonal IgG. The other glycoprotein type was labeled with fluorescence monovalent Fab' fragments that do not induce cross-linking, and its mobility was measured by FPR. Neither the mobile fraction nor the lateral diffusion coefficient of the Fab'-labeled viral glycoproteins was affected by immobilization of the second viral envelope protein, demonstrating that F and HN diffuse independently in the target cell membrane and are not associated in mutual complexes.