Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase beta 2 subunit

A reversible acid-denaturation process of the beta 2 subunit of Escherichia coli tryptophan synthase has been set up. The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured. The renaturation of this denatured state of beta 2 has been observed in a stopped-flow system, in the presence of a monoclonal antibody directed against native beta 2. It is shown that the association occurs very early in the folding of beta 2. The association rate constants of the antibody with the immunoreactive folding intermediate and with native beta 2 are the same (3 X 10(5) M-1.s-1). But at high antibody concentrations the formation of the antigen/antibody complex is rate limited by a rapid (5.4 X 10(-2) s-1) isomerization of refolding beta chains. This isomerization appears to reflect the formation of at least part of the epitope recognized by the antibody during the folding of beta 2. Further conformational adjustments occurring later in the folding pathway would then allow the ultimate structuring of the epitope.     

Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases

The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.     

Structure and dynamics of an acid-denatured protein G mutant

NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 degrees C, F30H-G(B1) is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding G(B1). We have assigned almost all backbone (15)N, H(N), and H(alpha) resonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both alpha-helical and beta-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for beta-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta1-beta2 hairpin of the folded state. Steady-state ?(1)H-(15)N? NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta3-beta4 hairpin, and the beta1-beta2 turn may serve as potential initiation sites for folding of G(B). Furthermore, residual structure in acid-denatured F30H-G(B1) is more extensive than in peptide fragments corresponding to the beta1-beta2, alpha-helix, and beta3-beta4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain.     

The free and the β2-microglobulin-associated heavy chains of HLA-A,B alloantigens share the antigenic determinant recognized by the monoclonal antibody Q1/28

Serological and immunochemical assays have shown that the monoclonal antibody Q1/28 recognizes an antigenic determinant which is expressed on the heavy chain of subsets of HLA-A, B antigens and is distinct from those defining the serological polymorphism of this system. Association of the HLA-A, B heavy chain with ₂-microglobulin is not required for expression of the antigenic determinant recognized by the monoclonal antibody Q1/28, since this antibody can immunoprecipitate a 45 000 m. w. component from radiolabeled lymphoid-cell glycoproteins immunodepleted with either an anti-human ₂-microglobulin xenoantiserum or the MoAb W6/32 to framework determinants of HLA-A, B, C antigens. Furthermore, the MoAb Q1/28 can immunoprecipitate a 45 000 m. w. component from an NP40lysate of radiolabeled Daudi cells, which lack the genetic information for ₂-microglobulin. The determinant recognized by the MoAb Q1/28 is relatively resistant to denaturing treatments and does not appear to be carbohydrate in nature. The MoAb Q1/28 is the first example of an antibody which recognizes an antigenic determinant expressed on both the ₂-microglobulin-associated and free HLA-A, B heavy chains.     

The Lex Carbohydrate Sequence Is Recognized by Antibody to L5, a Functional Antigen in Early Neural Development

Abstract: The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N -glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5-positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N -glycans containing a 3-fucosyl N -acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N -acetyllactosamine sequence [Lewis^x (Le^x) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the Le^x sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15.     

Kinetics and importance of the dimerization step in the folding pathway of the beta 2 subunit of Escherichia coli tryptophan synthase

During its folding, the polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (L-serine hydrolyase (adding indole) EC 4.2.1.20) undergoes dimerization. To decide whether this dimerization precedes or follows the formation of the native, functional, tertiary structure of the polypeptide chain, the kinetics of renaturation of beta 2 are studied by monitoring both the regain of native conformation and the dimerization. Dimer formation is followed by the increase of the fluorescence polarization, or by energy transfer between a fluorescence donor and a fluorescence acceptor, which occur upon association of adequately labelled beta chains. Renaturation is followed by the regain of functional properties of beta 2, i.e. its ability to bind pyridoxal-5'-phosphate or to form a fluorescent ternary complex with this coenzyme and L-serine. It is shown that for beta 2 the dimerization obeys first-order kinetics, presumably because it occurs rapidly after a rate-limiting isomerization of the monomer. The dimerization is followed by another isomerization, taking place within the dimer, which leads to the formation of the pyridoxal-5'-phosphate binding site. Still another, slow, isomerization reaction involving the F1 (N-terminal) domain completes the renaturation. With a modified form of beta 2 (trypsin-nicked beta 2) where this isomerization of F1 can be made to occur before the dimerization, the dimer is also shown to appear before the functional properties. It is concluded that a non-native dimer indeed exists as an obligatory intermediate on the folding pathway of nicked beta 2 and of beta 2, and that interdomain interactions are needed to force the polypeptide chains into their native conformations.     

The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea.

Shikimate kinase was chosen as a convenient representative example of the subclass of alpha/beta proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 m urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJ.mol-1. Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 m. The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 s-1) corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other alpha/beta domain proteins is discussed.     

Appearance of neoantigen in mouse IgG1 upon reduction of interchain disulfide bridges: assessment of local and general conformational rearrangements using monoclonal antibody and small-angle X-ray scattering

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)2 fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between CH and CL domains. The second produced monAb F6C2 reacts with CH1-CL region of reduced mouse IgG2. Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.     

An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.

The refolding kinetics of the tryptophan synthase beta 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured beta 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded beta 2, the fluorescence of ANS passes through a maximum after about 1 s and then 'slowly' decreases. These results show the accumulation, in the 1-10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the 'molten globule' state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5 raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶ - Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel -strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5.     

Appearance of Neoantigen in Mouse IgG1 Upon Reduction of Interchain Disulfide Bridges: Assessment of Local and General Conformational Rearrangements Using Monoclonal Antibody and Small-angle X-ray Scattering

Abstract

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)₂ fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between C_H and C_L domains. The second produced monAb F6C2 reacts with C_H1-C_L region of reduced mouse IgG2.

Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.     

Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins

Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.     

Identification of the DRw10 DR beta 1-chain allele as encoding a polymorphic class II major histocompatibility complex epitope otherwise restricted to DR beta 2 molecules of the DRw53 type

Although the polymorphic human Ia epitope recognized by monoclonal antibody 109d6 typically is expressed by DRw53 beta 2 chains, the epitope was shown to be encoded by distinctive DR beta 1 chains of a DRw10 haplotype in three unrelated DR4-negative individuals with rheumatoid arthritis. No evidence of a DR beta 2 (DR beta 4) chain molecule was found to be encoded by this haplotype. Using two-dimensional gel analysis and partial radioactive N-terminal microsequencing, the DR and DQ products were characterized in the heterozygous members of a family in which the segregation of both varieties of DR beta chains specifying the 109d6 epitope was demonstrated. The expression of the epitope on the DR beta 2 chain, but not on the DR beta 1 chain, was abolished by preventing N-linked glycosylation, although in both molecules the epitope was not altered by neuraminidase digestion. The potential structural bases of the serologic cross reactions of DRw10 are discussed, as are the possible implications of the findings for the definition of susceptibility to rheumatoid arthritis.     

Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked beta 1-2, beta 1-4, or beta 1-6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.     

Alternate succession of steps can lead to the folding of a multidomain oligomeric protein

The beta 2 subunit of Escherichia coli tryptophan synthase can be either unfolded in 6 M guanidine, or extensively denatured at acidic pH. These two denatured forms of beta 2 have different circular dichroism spectra and thus correspond to distinct physical states. Here we compare the folding pathways of these two different denatured forms of beta chains. We describe the kinetics of regain of a variety of physical, functional, and immunochemical signals characteristic of six successive steps previously identified on the folding pathway of guanidine unfolded beta 2. It is shown that whereas identical molecular events occur with the same kinetics, the two folding pathways are different, and involve different structural intermediates.     

The epsilon subunit and inhibitory monoclonal antibodies interact with the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase

The epitopes of two classes of monoclonal antibody and the binding site for the epsilon subunit have been mapped to the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase using partial CNBr cleavage, weak acid hydrolysis, and Western blots. One class of antibody, B-I, inhibits ATPase activity; the other class, B-II, recognizes an epitope not exposed on the surface of intact F1. Data from two-dimensional gels and blots of beta cleaved with CNBr/weak acid showed that the B-I epitope lies between Asp-381 and the carboxyl-terminal Leu-459, and the B-II epitope lies between Asp-345 and Met-380. Weak acid hydrolysis of the beta-epsilon product obtained by cross-linking F1 with a water-soluble carbodiimide yielded a fragment containing epsilon and a 13-kDa carboxyl-terminal fragment of beta indicating that epsilon interacts with this portion of beta as well. Fab fragments from the B-I antibody beta-6 could be cross-linked to the epsilon subunit in native F1 by various cross-linking agents demonstrating that the antibody and the epsilon subunit occupy adjacent, nonoverlapping sites on the beta subunit. Implications of these results for the roles of the epsilon subunit and of the carboxyl-terminal region of the beta subunit in F1 are discussed.     

Immunoreactivity between a monoclonal lupus autoantibody and the arginine/aspartic acid repeats within the U1-snRNP 70K autoantigen is conformationally restricted

Immunoreactivity of the arginine/aspartic acid (RD) repeats of the 70K protein of U1 small nuclear ribonucleoprotein (snRNP) was determined to be conformationally dependent. The monoclonal autoantibody 2.73, isolated from a lupus-prone MRL/n mouse model, is reactive with the RD repeat regions of U1 snRNP 70K protein. Immunochemical analysis of the antigenic determinants with use of chemically synthesized peptides characterized the 2.73 epitope as the RD repeat [Pelsue, S.,et al. (1993)Autoimmunity,15, 231–236] Analysis by circular dichroism (CD) and nuclear magnetic resonance spectroscopy indicates conformational preferences in the immunoreactive peptides. Computer analyses of CD spectra obtained on the RD-containing peptides predict-turns and-sheet to be the preferred conformations of the RD repeats. This structure was also predicted by the Chou-Fasman algorithm. The RD repeat is believed to be a conserved structural motif; however, the biological function is still unclear. Immunological and biochemical analysis of autoimmune antibodies and their respective antigenic determinants has helped to characterize the possible mechanisms that lead to autoimmune diseases. This is the first report of a conformationally dependent, linear epitope of an autoantibody.     

HLA-D gene studies in relation to immune responsiveness to a grass allergen Lol p III

The grass pollen allergen Lol p III (M _r11 000) is a well-characterized antigen that has been found useful in immunogenetic studies of human immune responsiveness. Since immune responsiveness to this allergen is associated with HLA-DR3, we investigated whether there was any sequence in the HLA-D region that would render a person susceptible [antibody (Ab)-positive] to the allergen. By sequence-specific oligonucleotide (SSO) slot-blot and sequence analyses of polymerase chain reaction (PCR)-amplified genomic DNA from Lol p III responder and nonresponder subjects, Ab responsiveness was found to be strongly associated with the sequence Glu-Tyr-Ser-Thr-Ser (EYSTS), present in the first polymorphic regions of DRI polypeptide chains of DR3, DR11 (split of DR5), and DRw6. Of the 41 grass-allergic subjects investigated, 19 had the EYSTS sequence, of whom 18 (95%) were Lol p III immunoglobulin G (IgG) Ab responder; among the 22 EYSTS^- subjects, ten were Lol p III responders (P=0.0001, relative risk=21.6). No such association was found with any polymorphic sequences in othe DR chains,or in DQI and DQI chains. These findings suggest that the EYSTS sequence is important in the presentation of an epitope of Lol p III; other sequence(s) may be involved in the presentation of othe epitope(s). To our knowledge, this is the first demonstration of a strong association between a specific HLA sequence and immune responsiveness to a well-defined antigen. The paper shows that presence of the EYSTS sequence classifies subjects as Lol p III responders in 18/19 cases.     

Strain variation and nuclear association of Newcastle disease virus matrix protein.

Five monoclonal antibodies to the matrix (M) protein of Newcastle disease virus (NDV) Australia-Victoria (AV) strain were generated and characterized. In competitive antibody-binding assays, the antibodies fell into three discrete groups. The antigenic sites described by these antibody groups were designated M1, M2, and M3. Each antibody reacted with a panel of NDV strains in a manner unique to its group, confirming the grouping by competitive antibody binding. Only site M1 was found on all 12 of the strains tested and may be a "pan-NDV" epitope. A large portion of the M protein of strain AV was detected in the nuclei of infected cells by all five monoclonal antibodies. In addition, the antibodies only stained the nuclei of cells infected with NDV strains expressing M protein containing the corresponding antigenic site. These results confirm that the immunoreactivity in the nucleus is actually caused by the M protein and not by a cross-reacting host protein induced by viral infection.