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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.83, #1-3)

Superantibodies by Heinz Kohler (pp. 1-12).

The antibody molecule possesses a number of so-called unconventional binding sites in the variable domain that are expressed and function independently from the antigen-binding site. These sites are encoded in the germiline, predominantly in framework residues. By this definition, these sites function as part of the innate immunity, and are not subject to antigendriven mutation and maturation. In this article, we focus on the evidence for the function and utility of the self-binding domain. The self-binding or autophilic domain has been discovered on murine germline-encoded antibodies from the S107/T15 Vh family. Autophilic antibodies form self-complexes after attaching to targets, but remain monomeric in solution. A peptide has been identified that confers self-binding if chemically attached to antibodies. Because this modification enhances the overall avidity of antibodies for target binding, therapeutic and diagnostic antibodies can be biotechnologically improved.The concept of super antibodies is introduced here to describe the unique coexistence and synergism of acquired immunity with innate immunity via antigen-specific and unconventional functional domains. As not every antibody qualifies as a super antibody, biotechnology engineering can produce superantibodies with superior targeting and therapeutic properties.

Keywords: Antibody; variable domain; unconventional binding site; autophilic binding; superantibody

Tracking immunoglobulin variable-gene expression in HIV infection by Moncef Zouali (pp. 13-29).

B-cell SAgs interact with normal human nonimmune immunoglobulins (Igs) independently of the light-chain isotype, and activate a large proportion of the B-cell repertoire. Recently, the major envelope protein of human immunodeficiency virus 1 (HIV-1), gp120, was found to exhibit SAg-like properties for B cells with potential pathological consequences for the infected host, including accelerated apoptosis and progressive loss of B cells. This unconventional mode of interaction contrasts with its binding to immunization-induced antibodies, which requires the tertiary structure of the heavy- and light-chain variable regions. Examining the temporal development of V_H3⁺ antibodies in HIV-1-infected subjects over a 7-yrperiodshowed that V_H3⁺ antibodies specific for the gp 120 SAg-binding site are deficient. Quantification of V_H3⁺ antibodies, which impart protective responses to infectious agents, in serum samples from HIV-seropositive slow progressors and from patients who progressed to AIDS-related manifestations reveals that paucity in V_H3⁺ antibodies is a marker of rapid clinical decline. Remarkably, anti-gp 160 V_H3⁺ antibodies show a gradual decrease in progressors and vary with time, depending on the viral load. Thus, V_H3⁺ antibodies could play an importantrole in protection, and their underexpression may accelerate disease progression. Investigation of the structural basis of the interaction between human Igs and gp 120 shows that the viral gp 120 SAg can interact only with a subset of human V_H3⁺ Igs. A number of amino acid-positions present primarily in the first and third framework regions of the Ig heavychain variable regions correlate with gp 120 binding. These residues partially overlap with the Staphylococcus aureus protein A-binding site for V_H3⁺ Igs. Overall, these interactions could represent a novel mechanism of humoral deficiency resulting from the capacity of a viral SAg to impact an important subset of the B-cell repertoire and to induce B-cell death by apoptosis.

Keywords: Superantigen; gp 120; HIV; B cell; human antibodies; immunoglobulin variable genes

Epitope promiscuity of human monoclonal autoantibodies to T-cell receptor-combining site determinants by John J. Marchalonis; Ian Robey; Samuel F. Schluter; David E. Yocum (pp. 31-52).

To characterize the binding specificity and light- and heavy-chain variable region usage in monoclonal human autoantibodies (mAAbs) to T-cell receptors, we constructed heterohybridomas from peripheral blood B cells of three rheumatoid arthritis (RA) patients. From a panel of more than 200 heterohybridomas secreting IgM autoantibodies binding to T-cell receptor Vß chain first complementarity determining segments (CDR1), we characterized two IgM/λ molecules from a single patient in detail. These bound to both CDR1 peptide epitopes and intact TCR of recombinant single-chain T-cell receptor constructs, and to T-cell surface TCR. Spectratype analysis using epitopes mimicking a set of 24 Vß genes indicated that one molecule bound only a few members of the set, whereas the second showed considerable epitope promiscuity by binding to more than half of the tested CDR1 peptides. Both mAAbs used variants of a Vλ3 gene that were very similar to one another and to the germline gene. The epitope-promiscuous autoantibody used a V_H4 gene identical to a germline prototype, while the other incorporated a V_H3 sequence differeing in only a single residue from its germline prototype. The CDR3s of both were large and distinct from each other as well as from the corresponding segments of rheumatoid factors and “cold agglutinins” using the same or related V_H germline genes. These mAAbs offer models for deciphering the basis of epitope promiscuity, and serve as candidates for direct use in immunomodulation because they are of intrinsic human origin and do not require molecular engineering to adapt them for use in therapy.

Diversification and selection mechanisms for the production of protein repertoires by M. S. Neuberger; J. E. Sale; S. J. Cumbers; C. J. Jolly; M. P. Bemark; M. R. Ehrenstein; A. Lanoue; M. Brüggemann; F. D. Batista; S. L. Davies; G. T. Williams (pp. 53-62).

The physiological mechanism for producing antigen-specific antibodies is based on a two-phase neo-Darwinian process: the first phase consists of diversity generation (formation of the repertoire), and the second phase is antigen-mediated selection. In this article, we consider how the natural immunoglobulin gene-diversification processes can be exploited both in vivo and in vitro in order to allow the generation of novel antibody (and heterologous protein) repertoires.

Keywords: Rerarrangement; somatic hypermutation; antibody; immunoglobulin; transgenic mouse

Structural components responsible for peptide antigenicity by Michael Sela (pp. 63-70).

The conformation of a peptide defines its antigenic specificity. In most cases, a free peptide is in a random form, whereas the same peptide within a protein hasa defined conformation. Immunological cross-reactions between the two are rare. Nevertheless, there are cases where an anticonformational antibody may transconform (change the conformation of) the free peptide, allowing the reaction to occur.Based on such considerations, synthetic vaccines against infectious diseases may be constructed. The same is true for autoimmune diseases, where, at least in one case—that of the exacerbating-remitting type of multiple sclerosis— we have developed a polypeptidic drug-vaccine, copolymer 1 (denoted Copaxone), capable of suppressing the autoimmune phenomena in a specific way. This drug-vaccine has been approved for treatment by the US Food and Drug Administration (FDA).

Keywords: Copolymer 1; multiple sclerosis; immuogenicity; conformation

Natural catalytic immunity is not restricted to autoantigenic substrates by Sudhir Paul; Ravi S. Kalaga; G. Gololobov; Douglas Brenneman (pp. 71-84).

The autoimmune repertoire is well known from previous studies to be capable of producing catalytic antibodies directed to self-antigens. In the present study, we explored the ability of 26 monoclonal light chains (Lchains) from multiple myeloma patients to cleave radiolabeled gp 120, a foreign protein. One L chain with this activity was identified. ¹²⁵I-gp120 and unlabeled gp 120 were cleaved at several sites by the L chain, as shown by SDS-polyacrylamide gel electrophoresis, autoradiography, and immunoblotting, respectively. The apparent dissociation constant of the L chain was 130–145 nM, indicating high-affinity gp 120 recognition. ¹²⁵I-albumin was not cleaved by the L chain, and various proteins and peptides did not inhibit gp 120 cleavage by the L chain, suggesting that the activity is not a nonspecific phenomenon. The substrate recognition determinants may be conserved in different HIV-1 strains, because gp 120 isolated from strains SF2, MN, and IIIB was found to be cleaved by the L chain. Micromolar concentrations of a synthetic peptide corresponding to residues 23–30 of gp 120 inhibited the cleavage of ¹²⁵I-gp 120, suggesting that these residues are components of the epitope recognized by the L chain. The toxic effect of gp120 in neuronal cultures was reduced by about 100-fold by pretreatment of the protein with the L chain. These observations open the possibility of utilizing gp120-cleaving antibodies in the treatment of AIDS.

Keywords: HIV-1; AIDS; gp 120; catalytic antibody; light chain

Does catalytic activity of bence-jones proteins contribute to the pathogenesis of multiple myeloma? by Hyogo Sinohara; Kinji Matsuura (pp. 85-94).

Some Bence-Jones proteins have been found to be capable of hydrolyzing DNA, chromogenic amide substrates, such as benzoylarginine p-nitroanilide, and natural oligopeptides, such as arginine vasopressin. Patients who excrete Bence-Jones protein with the DNA-nicking activity have shown moderately severe symptoms. When incubated with LLC-PK₁ (porcine kidney proximal tu bule) cells, some Bence Jones proteins penetrated the cytoplasm, and en tered the nucleus with little or no degradation of epitopes. Intranuclear Bence Jones proteins sultimately induced DNA fragmentation in situ and cell death. This cytocidal activity was not directly associated with the DNA-nicking activity, since Bence Jones proteins with no detectable DNase activity also produced cell death. These results, however, suggest that the biological activities of Bence Jones proteins described here makes a significant contribution to the development and/or deterioration of multiple myeloma.

Keywords: Bence Jones proteins; cytotoxicity; DNase activity; LLCLK₁ cell line; peptidase activity; nuclear import

DNA hydrolysis by monoclonal autoantibody BV 04-01 by L. S. Rodkey; G. Gololobov; C. A. Rumbley; J. Rumbley; D. V. Schourov; O. I. Makarevich; A. G. Gabibov; E. W. Voss Jr (pp. 95-105).

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg²⁺. Catalysis was asssociated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A₇C₇ATATAGCGCGT₂, as well as a preference for cleaving within CG-rich regions of dsDNA. Data nn specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg²⁺ coordination site composed of the L32Tyr and L27d His side chains and a DNA 3′-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V_H and V_L genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important—rarely examined—function for some antibody molecules.

Keywords: Catalysis; autoantibody; abzyme

Amidase and peptidase activities of polyclonal immunoglobulin G present in the sera of patients with rheumatoid arthritis by Kinji Matsuura; Shinya Ikoma; Masafumi Sugiyama; Masanori Funauchi; Hyogo Sinohara (pp. 107-114).

Polyclonal Immunoglobulin (Ig) G from patients with rheumatoid arthritis (RA) and healthy subjects hydrolyzed carbobenzoxy−Val−Gly−Arg p-nitroanilide and D−Pro−Phe−Arg p-nitroanilide. RA IgG exhibited higher activity against the former substrate, but not the latter. On the other hand, RA IgG showed reduced activity against D−Pro−Phe−Arg methylcoumarinamide, when compared with those of the healthy controls. These results suggest that RA IgGs differ from normal IgGs in the substrate specificity of amidase activity. Preliminary studies have shown that two out of three RA IgG samples cleaved a pentapeptide—Gln−Arg−Arg−Arg−Ala−Ala— which is assumed to be associated with the risk of developing RA (Gregersen, P. K. et al. (1987), Arthritis Rheum. 30, 1205–1213). By contrast, virtually no cleavage of the same peptide was observed with IgG from healthy controls. A peptide analog, Gln−Arg−Arg−Trp−Ala, was not cleaved at all by any IgGs examined either from RA patients or healthy controls.

Keywords: Amidase activity of IgG; peptidase activity of IgG; rheumatoid arthritis; shared epitope

Secretory immunoglobulin a from health y human mothers' milk catalyzes nucleic acid hydrolysis by Georgy A. Nevinsky; Tat'yana G. Kanyshkova; Dmitry V. Semenov; Alexander V. Vlassov; Anastasiya V. Gal'vita; Valentina N. Buneva (pp. 115-130).

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.

Keywords: Human milk; sIgA; catalytic antibodies; DNA- and RNA-hydrolyzing activities

Localization of a novel adhesion-promoting site on acetylcholinesterase using catalytic antiacetylcholinesterase antibodies displaying cholinesterase-like activity by Glynis Johnson; Samuel W. Moore (pp. 131-144).

A monoclonal antibody (MAb) raised against human acetylcholinesterase was found to have catalytic activity. A similar phenomenon was observed in a polyclonal antibody raised against the same antigen. The antibodies were demonstrated to be pure, and no contamination with either acetylcholinesterase or butyrylcholinesterase was found. Both antibodies hydrolyzed acetylthiocholine, anacetylcholinesterasesubstrate, and the MAb followed Michaelis-Menten kinetics. Six other MAbs and one other polyclonal antibody showed no evidence of catalytic activity.Acetylcholinesterase is a key component in the transmission of the nerve impulse, and is also expressed nonsynaptically during embryonic development, and abnormalities in expression are seen in neural tumors and degenerative disorders. This unusual expression is believed to be associated with a novel function of the enzyme related to differentiation and cell adhesion. Autoantibodies to acetylcholinesterase have been observed in a variety of neurologic, muscular, and autoimmune disorders.In an investigation of the possible role of acetylcholinesterase in cell adhesion, we showed that the enzyme promoted neurite outgrowth, in neuroblastoma cell lines, and conversely, that certain antiacetylcolinesterase antibodies abrogated cell-substrate adhesion. Interestingly, the antibodies most effective in this regard were catalytic. Preliminary epitopeanalysis indicated a conformational epitope in the N-terminal domain. This domain contains the active site, within a deep gorge, and the peripheral anionic site at the rim of the gorge. Peripheral-site inhibitors, but not active-site inhibitors, also interfered with adhesion, and competed with the catalytic monoclonal binding to acetylcholinesterase, indicating that the epitope recognized is associated with the peripheral anionic site. The inhibitor data also support the supposition that catalysis in these antibodies may have arisen from stable complexation of acetylcholinesterase with an inhibitor.We conclude that the catalytic antiacetylcholinesterase antibody interacts with structures associated with the peripheral anionic site, thus defining a novel site on the molecule involved, in cell adhesion. This finding has implications for our understanding of the potential importance of this peripheral site in a variety of congenital, neoplastic, and degenerative conditions.

Keywords: Catalytic antibody; acetylcholinesterase; cell adhesion; peripheral anionic site

General discussion I by D. Capra (pp. 145-153).

Idiotypic network dysregulation by Yaniv Sherer; Yehuda Shoenfeld (pp. 155-162).

The pathogenesis of autoimmune disease is still an enigma. Whereas the diverse clinical manifestations of many autoimmune diseases cannot be explained by the existence of autoantibodies, idiotypic dysregulation may provide an alternative explanation. Experimental models, serum level changes of pathogenic idiotypes during exacerbation and remission, and the increased expression of pathogenic idiotypes following common infections all support this notion. In this article we review experimental models of autoimmune disease induction (systemic lupus erythematosus, antiphospholipidsyn drome, Goodpasture's syndrome, autoimmune thyroiditis, and vasculitis) by manipulation of the idiotypic network and discuss the utilization of idiotypes for the immunotherapy of autoimmune diseases and other conditions that involve the immune system (e.g., atherosclerosis).

Keywords: Autoimmunity; idiotypes; systemic lupus erythematosus

Catalytic mechanism of an abzyme displaying a beta-lactamase-like activity by B. Avalle; H. Débat; A. Friboulet; D. Thomas (pp. 163-171).

A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site. We have previously demonstrated that this IgG has retained some of the structural information displayed by the beta-lactamase active site, evident from data that polyclonal anti-Ab2 antibodies (Ab3) recognize beta-lactamase. In this article, we investigated the catalytic mechanisms of the abzyme compared to that of the enzyme. The experimental data, allow us to draw hypothesize the catalytic residues required for catalysis.

Keywords: Beta-lactamase; antiidiotypic antibody; abzyme; catalytic antibody; suicide substrate

Nonspecific catalysis by protein surfaces by Anthony J. Kirby; Florian Hollfelder; Dan S. Tawfik (pp. 173-181).

Catalytic antibodies are the best availablea llaround enzyme mimics. They provide a unique experimental approach and some special insights into general questions about catalysis by enzymes. They offer enantiospecific reactions and levels of substrate binding that compare well with typical enzyme reactions, but not—so far—comparable catalytic efficiency. We and others have used the Kemp elimination as a probe of catalytic efficiency in antibodies. We compare these reactions with nonspecific catalysis by other proteins, and with catalysis by enzymes. Several simple reactions are catalyzed by theserum albumins with Michaelis-Menten kinetics, and can be shown to involve substrate binding and catalysis by local functional groups. Here, we report the details of one investigation, which implicate known binding sites on the protein surface and discuss implications for catalyst design and efficiency.

Keywords: Antibodies; catalysis; transition-state analog; albumin; BSA

Pyridoxal-5′-phosphate-dependent catalytic antibodies by Svetlana I. Gramatikova; Philipp Christen (pp. 183-193).

Cofactors—i.e., metal ions and coenzymes—extend the catalytic scope of enzymes and might have been among the first biological catalysts. They may be expected to efficiently extend the catalytic potential of antibodies. Monoclonal antibodies (MAbs) against N^α-phosphopyridoxyl-l-lysine were screened for 1) binding of 5′-phosphopyridoxyl amino acids, 2) binding of the planar Schiff base of pyridoxal-5′-phosphate (PLP) and amino acids, the first intermediate of all PLP-dependent reactions, and 3), catalysis of the PLP-dependent α, β-elimination reaction with β-chloro-D/L-alanine. Antibody 15A9 fulfilled all criteria and was also found to catalyze the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k′ _cat=0.42 min⁻¹ with D-alanine at 25°C). No other reactions with either D- or L-amino acids were detected. PLP markedly contributes to catalytic effecacy—it is a 10⁴ times more efficient acceptor of the amino group than pyruvate. The antibody ensures reaction specificity, stereospecificity, and substrate specificity, and further accelerates the transamination reaction (k′ _cat(Ab)/k′ _cat(PLP)=5×10³). The successive screening steps simulate the selection criteria that might have been operative in the evolution of protein-assisted psyridoxal catalysis.

Keywords: Catalytic antibodies; pyridoxal-5′-phosphate; transamination

Catalytic antibodies for complex reactions by Binodh S. DeSilva; György Orosz; Kamal L. Egodage; Robert G. Carlson; Richard L. Schowen; George S. Wilson (pp. 195-208).

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanideion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity atan early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in deriving one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.

Keywords: Catalytic antibody; transition-state analog; NDA/CN reaction; screening; enzyme mimics

How and why 41S-2 antibody subunits acquire the ability to catalyze decomposition of the conserved sequence of gp41 of HIV-1 by Emi Hifumi; Yoshiko Okamoto; Taizo Uda (pp. 209-220).

It has become well known that antibodies obtained by immunization with the ground state of peptides can display proteolytic activity. Our antibody light chain produced by immunization with the peptide RGP DRPEGIEEEG-GERDRD, a highly conserved sequence in envelope gp41 of HIV-1 showed the ability to cleave this peptide. Moreover, its heavy chain also decomposed the peptide, although this occurred at lower activity levels compared with the light chain, while the whole antibody did not show any catalytic activity. From molecular modeling, the light and heavy chains of the antibody were deduced to possess catalytic triads (Asp, His, and Ser) in their steric conformations, which may be responsible for the observed proteolytic activity.

Keywords: Catalyticativity of 41S-2 antibody to HIV-1 envelope gp41; catalytic antibody; HIV; gp41; conserved sequence

Nucleophilic proteolytic antibodies by Gennady Gololobov; Alfonso Tramontano; Sudhir Paul (pp. 221-232).

Proteolytic antibodies appear to utilizecatalytic mechanisms akin to nonantibody serine proteases, assessed from mutagenesis and protease-inhibitor studies. The catalytic efficiency derives substantially from the ability to recognize the ground state with high affinity. Because the proteolytic activity is germline-encoded, catalysts with specificity for virtually any target polypeptide could potentially be developed by applying appropriate immunogens and selection strategies. Analysis of transition-state stabilizing interactions suggests that chemical reactivity ofactive-site serine residues is an important contributor to catalysis. A prototype antigen analog capable of reacting covalently with nucleophilic serine residues permitted enrichment of the catalysts from a phage-displayed lupus light-chain library. Further mechanistic developments in understanding proteolytic antibodies may lead to the isolation of catalysts suitable for passive immunotherapy of major diseases, and elicitation of catalytic immunity as a component of prophylactic vaccination.

Keywords: Catalytic antibodies; phage display; serine proteases

Inhibition and labeling of enzymes and abzymes by phosphonate diesters by A. Tramontano; B. Ivanov; G. Gololobov; S. Paul (pp. 233-243).

Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymaticactivity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of try psin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permitanalysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDSPAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretretment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.

Autoantibodies to thyroglobulin in health and disease by Noel R. Rose; C. Lynne Burek (pp. 245-254).

Thyroglobulin (Tg)—a heavily glycosylated, iodinated protein—isa major autoantigen in autoimmune thyroiditis. Tg also induces thyroiditis by immunization of experimental animals. Humans with chronic lymphocytic thyroiditis characteristically produce autoantibodies to thyroglobu lin, but similar autoantibodies are also found in some clinically normal, euthyroid individuals. A comparison of the fine specificity of autoantibodies in humans and in experimentally immunized mice was carried out, based on their ability to inhibit a panel of monoclonal antibodies (MAbs). Patients with autoimmune thyroid disease, as well as normal individuals, produced autoantibodies mainly to the conserved, cross-reactive determinants of thyroglobulin. Patients developed additional autoantibodies to species-restricted epitopes. The determinants recognized by patients with Graves' disease differed in some respects from epitopes recognized by thyroiditis patients or patients with differentiated thyroid carcinoma. Similarly, mice that are genetically susceptible to thyroiditis produced autoantibodies that reacted with the mouse-specific antigenic determinants. Using an autoantibody that reacts with one of the epitopes associated with thyroiditis, a reactive 15-k Da fragment of human Tg—localized at the carboxy end of the molecule—was isolated and sequenced. Iodine plays an important role in the precise specificity of the disease-associated epitope, since T cells from patients with thyroiditis react with iodinated but not noniodinated human thyroglobulin. Addition of iodine to Tg generates new or cryptic epitopes. Use of a selected MAb as a surrogate for the T-cell receptor suggests that a specific iodine-containing epitope is sometimes involved in recognition. Finally, thyroglobulin-reactive autoantibodies exhibit proteolytic activity on thyroglobulin.

Keywords: Thyroglobulin; iodine; thyroiditis; autoimmune disease; autoimmunity; autoantigen; thyroxine

Autoantibodies to nuclear antigens by A. V. Kozyr; A. V. Kolesnikov; N. A. Zelenova; L. P. Sashchenko; S. V. Mikhalap; M. E. Bulina; A. N. Ignatova; P. V. Favorov; A. G. Gabibov (pp. 255-269).

Thecytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10⁻¹⁰ M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma, and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA). Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity of cytotoxic anti-DNA antibodies are discussed in this article.

Keywords: Anti-DNA antibodies; catalytic autoantibodies; cytotoxicity; nuclear antigens

Multifunctional antigens of A. fumigatus and specific antibodies by Swagata Purkayastha; Taruna Madan; Ashok Shah; H. G. Krishnamurthy; P. Usha Sarma (pp. 271-286).

The majority of Aspergillus-induced infections in man are caused by the pathogenic fungus A. fumigatus, which secretes biologically and immunologically active glycosylated and nonglycosylated proteins. The complexity in the antigenic structure of A. fumigatus and the varying host immune responses lead toa widespectrum of clinical conditionssuch as allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and invasive aspergillosis. It is reported that 15–20% of allergic asthmatics suffer from Aspergillus-induced allergies. The incidence of opportunistic infections, including Aspergillus infections, has risen because of the increase in the incidence of HIV and tuberculosis. Allergic bronchopulmonary aspergillosis is an immunologically significant clinical form where type land type III hypersensitivity reactions are involved in pathogen esis. High levels of specific IgE and IgG antibodies in these patients are of diagnostic value. Molecular characterization of certain immunodominant all ergens and antigens of A. fumigatus revealed the presence of complex carbohydrate moieties, heat-shock proteins, enzyme activities such as elastase, protease, catalase, dismutase, and cytotoxic ribonuclease. A Con A binding allergen/antigen (45k Da) and Con Anonbinding allergen/antigen (18 kDa, Asp fI) have a multifunctional nature. The multifunctional nature of these antigens may play an important role in the pathogenesis of the disease. Significant amounts of a major allergen/antigen of molecular weight 18 kD a is excreted in large amounts through the urine of patients with invasive aspergillosis. Studies on the structure—function relationship of the 18-kDa allergen/antigen revealed the involvement of tryptophan residues in binding with monoclonal antibodies (MAbs). Also, the histidine residues and cysteine disulfide, bonds of the 18-kDa allergen are involved in its catalytic activity. The high load of multifunctional antigens in the serum of patients for prolonged periods, the presence of high levels of specific antibodies, and the absence of protective antibodies in ABPA patients have necessitated studies on the functional properties, of the antibodies. The present study shows significant immunoreactivity of antibodies in patients of ABPA to fibronectin and collagen Analysis of IgG antibodies from the patients of ABPA showed the presence of DNA-cleaving activity. These observations offer a new line of thinking in understanding the mechanism of pathogenesis of Aspergillus-induced clinical manifestations, and may lead to novel approaches to intervention in the inflammation and infection caused by fungal pathogens.

Keywords: Allergic bronchopulmonary aspergillosis (ABPA); Aspergillus fumigatus ; ribonuclease; protease; fibronectin; collagen type IV; DNA cleaving activity

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods by Elsa Nedonchelle; Olivier Pitiot; Mookambeswaran A. Vijayalakshmi (pp. 287-295).

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.

Keywords: Catalytic autoimmune antibodies; activity preservation; immobilized histidine; affinity chromatography

General discussion II by S. Paul (pp. 297-313).

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.83, #1-3)