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D E Isenman

Publications and source records attributed to D E Isenman.

49 records · Page 3Linked to original sources
PubMed1981

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

Anilino Naphthalenesulfonates↗
PubMed1981

Expression of biological effector functions by immunoglobulin G molecules lacking the hinge region.

Several biological effector functions mediated by sites on the Fc region of human IgG1 have been studied in two variant IgG1 kappa monoclonal proteins (Dob and Lec) which contain deletions corresponding to the entire hinge region of the heavy chains. Neither Dob nor Lec protein in aggregated form was able to activate the classical complement pathway, and this was shown to be due to an inability to bind the first component of complement (C1). By rosette inhibition assays, Dob and Lec proteins were shown to have no measurable affinity for Fc receptors on human B cells or neutrophils. Dob and Lec proteins had a much reduced affinity for Fc receptors on the murine macrophage-like cell line P388D1 when compared to normal human IgG1. Furthermore, the hinge-deleted proteins were able to compete with murine IgG2b for P388D1 receptors but not with murine IgG2a. In contrast, the binding of Dob and Lec proteins to protein A from Staphylococcus aureus was entirely normal. The functional consequences of the hinge deletion were parallel to those seen when normal IgG1 was reduced and alkylated. It was concluded that the functional impotency of Dob and Lec proteins was related to the close association between the Fab and Fc regions in these molecules and the limited degree of segmental flexibility permitted in the absence of the hinge region. The data also suggest a major role for the C gamma 2 domain (C is the constant region) in mediating effector functions in normal IgG1.

Animals↗
PubMed1980

The interaction of C5 with C3b in free solution: a sufficient condition for cleavage by a fluid phase C3/C5 convertase.

We have measured the interactions of C3b with C5 in free solution under conditions that favor detecting weak binding interactions (high C3b and low C5 concentrations and low ionic strength). When a mixture of 125I-C5 (2 X 10(-8) M) and unlabeled C3b (3.8 x 10(-5) M) was ultracentrifuged in a sucrose gradient, virtually all of the C5 sedimented to the position of a 13 to 14S complex. In contrast, a sedimentation rate for C5 of 9S was obtained in the absence of C3b. The ability to bind C5 was observed to be a property of C3b since native C3 was unable to bind C5. It was also found that beta 1H by itself could inhibit the binding of C5 to cell-bound C3b. From inhibition studies, we estimate that the association constant for the C3b-C5 interaction is on the order of 2 x 10(6) M-1 in a low ionic strength buffer (mu = 0.06) and 5-fold weaker at physiologic ionic strength. C5 bound to C3b in free solution was cleaved by nephritic factor-stabilized fluid phase C3bB. C5 activation did not occur on omitting C3b. We conclude that the ability to bind C5 is a property of C3b molecules whether surface bound or in free solution and that when C5 is bound in either fashion, it can be cleaved by a fluid phase C3/C5 convertase.

Animals↗
PubMed1980

The effects of mild reduction on the structure and function of C3.

Reduction and alkylation of C3 in nondenaturing buffers by 2 mM DTT (1 hr, 23 degrees C) (C3 R/A) results in a > 99% loss of C3 hemolytic activity. In contrast, sham alkylated protein, or protein allowed to reoxidize from the mildly reduced state, showed full activity. The loss in lytic activity correlated with the alkylation of approximately 6 liberated sulfhydryls, all of which arose from intrachain disulfides in the alpha polypeptide chain of C3. Data from CD experiments indicate that these bonds are essential for maintaining the native conformation of the protein. The loss of hemolytic activity of C3 R/A was neither due to its inability to be cleaved by C3 convertases nor to a dysfunction of the cleavage-induced labile membrane binding site as cells bearing C3 convertases firmly bound C3 R/A to an extent 80% that of native C3. The hemolytic defect in C3 R/A was found to result from its inability, once deposited on C42-bearing cells, to bind C5 and thereby participate in the formation of a C5 convertase. Unlike the effect on hemolytic activity, C3 R/A showed only a partial loss (3-fold) in its ability to bind to C3 receptors on monocytes. Taken together, these studies differentiate in molecular terms several of the functional sites in C3 and stress the significance of intra-alpha-chain disulfides in maintaining the conformation in activated C3 required for binding of C5.

Binding Sites↗
PubMed1979

Folding pathways of immunoglobulin domains. The folding kinetics of the Cgamma3 domain of human IgG1.

The in vitro folding kinetics of a fragment corresponding to an intact dimer of the Cgamma3 domain of human IgG1 (pFc') were monitored via the large changes in tryptophan fluorescence which accompany these processes. In going from the guanidine hydrochloride (Gdn.HCl) induced unfolded state (4.0 M Gdn.HCl) to the native state (0.5 M Gdn.HCl), three well-separated first-order processes were observed having time constants of 5, 50, and 350 s and roughly equal amplitudes. These values were concentration independent, a fact consistent with there being no fluorescence change accompanying dimerization. These time constants are one to two orders of magnitude slower than those observed for proteins of similar size such as ribonuclease or cytochrome c, most probably reflecting the complex processes involved in forming the correct beta-sheet arrangement of immunoglobulin domains. The corresponding unfolding transition is biphasic having time constant values of 50 and 500 s, the latter comprising 80% of the fluorescence change. These data indicate the presence of at least one species with intermediate fluorescence along the unfolding pathway. Gdn.HCl concentration jumps were also performed over various intervals within the transition zone. The results are not consistent with a fully reversible mechanism. In the absence of the intrachain disulfide bond, pFc' exists in an unfolded state even at 0.5 M Gdn.HCl. In a concomitant refolding and reoxidation experiment (at 0.5 M Gdn.HCl and using an optimal disulfide interchange catalytic system), the time constant for disulfide formation was in the range of 80--200 s and the fluorescence change revealed a lag phase analyzable in terms of rate-limiting reoxidation and refolding times consistent with those observed for the initially disulfide bonded species. Under similar conditions but a 4 M Gdn.HCl, reoxidation was more than two orders of magnitude slower, suggesting that reoxidation is directed by a refolding nucleation event.

Disulfides↗
PubMed1977

Correlation between the exposure of aromatic chromophores at the surface of the Fc domains of immunoglobulin G and their ability to bind complement.

The recognition that certain biological effector functions associated with the Fc region of human IgG are mediated exclusively by either the Cgamma2 or Cgamma3 domains prompted a study of some of the physical properties of the isolated domains in an attempt to correlate these with functional differentiation. The degree of aromatic chromophore exposure of intact Fc and fragments corresponding to the Cgamma2 and Cgamma3 domains were determined by solvent perturbation difference spectroscopy using 20% ethylene glycol. For the monomeric Cgamma2 fragment one of the two tryptophans and all four of the tyrosines were exposed to solvent. In the pFc' fragment, which represented a dimer of two intact Cgamma3 domains, an average of 0.4 of the two tryptophans of 3.3 of the five tyrosines per chain were exposed. These data were consistent with the suggested involvement of tryptophan in complement fixation since Cgamma2 binds C1q but pFc' does not. Several fragments derived from the Cgamma3 region had previously been shown to have differing environments for their aromatic side chains from circular dichroism studies. These fragments have now been shown to exhibit different degrees of chromophore exposure to solvent. Removal of the carboxy-termimal heptapeptide from the intact, Cgamma3 domain resulted in a fragment not only showing a greater exposure of aromatic residues but also having the ability to bind Clq. Our data suggest that the structural requirements for C1Q binding may be quite commonplace within Fc, but tertiary folding limits their expression except in Cgamma2 in the native molecule. The solvent perturbation observed with Fc was somewhat lower than would have been expected from the results with the isolated domains, suggesting that interdomain interactions may result in burial of aromatic residues.

Binding Sites↗
PubMed1975

The structure and function of immunoglobulin domains: studies with beta-2-microglobulin on the role of the intrachain disulfide bond.

Beta-Microglobulin, a low-molecular-weight protein structurally related to the homology regions of immunoglobulins, has been used to study the role of the intrachain disulfide bond in the unfolding of immunoglobulin domains. The intact protein could be reversibly unfolded in guanidine hydrochloride, as judged by circular dichroism and optical rotation. Similarly, reoxidation of the reduced protein, during transfer from high concentrations of guanidine to neutral aqueous buffer, yielded a product with spectral characteristics typical of the native protein. However, if the free SH groups were prevented from reoxidizing either by chemical modification or by holding them in the reduced state, the molecule appeared to be in the randomly coiled state even under conditions where the intact protein is in the native conformation, judged on the basis of chiroptical measurements. The complement-fixing activity exhibited by native beta-2-microglobulin was retained by the reduced and alkylated derivative, suggesting that the site may be formed from a linear array of amino acids. We suggest a model for the folding of beta-2-microglobulin (and immunoglobulin domains) in which one of the early folding events results in disulfide bond formation, the latter being an obligatory step for continued folding to the native state.

Beta-Globulins↗
PubMed1975

The structure and function of immunoglobulin domains. II. The importance of interchain disulfide bonds and the possible role of molecular flexibility in the interaction between immunoglobulin G and complement.

The observation that reduction of the inter-chain disulfides in rabbit antibody destroys its ability to interact with complement was confirmed and shown to be true also of human meyloma IgG1 subclass proteins. In the latter case a C1-binding assay was used. Further studies indicated that it was the interheavy chain disulfides which were essential for complement-binding activity: Non-covalently reassembled IgG (LHHL) was devoid of C1-fixing activity whereas molecules formed from covalently linked heavy chain dimers, and reduced and alkylated light chains (ie., LH-HL) were as active as the parent intact IgG. Fc fragments from IgG1 bound C1 and this activity was insensitive to the presence or absence of intact interchain disulfides. These bonds therefore are neither directly involved in C1 binding nor essential for the integrity of the binding site. We have also shown that although IgG4 does not bind C1, Fc fragments derived from this subclass fix C1 with an affinity comparable to that of the corresponding fragment from IgG1. These data suggest that quaternary interaction with other regions of the molecule (ie., Fab) may modulate the activity of the C1-binding site.

Alkylation↗
PubMedDate not supplied

The use of diafiltration for the measurement of dialysable ligand binding to macromolecules: an examination of the theoretical parameters using concanavalin A in an Amicon cell.

The technique of diafiltration as a means of determining the characteristics of small ligand binding by proteins has been evaluated by measuring the binding of 45Ca to concanavalin A. Deposition of protein on the diafiltration membrane was found to be an important source of error but which could be eliminated by presaturation of the membrane with polylysine. The association constant for concanavalin A and calcium was 1.8 x 10(4) M-1, with a value of n = 2.2 atoms of calcium being bound per dimer of concanavalin A. The concentration of protein relative to the dissociation constant is also an important variable, and it was found that the method of diafiltration is only useful when protein concentrations greater than the value of the dissociation constant are used.

Calcium↗