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D E Isenman

Publications and source records attributed to D E Isenman.

At least 37 records · Page 2Linked to original sources
PubMed1992

Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18).

The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.

Amino Acid Sequence↗
PubMed1991

Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.

To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.

Amino Acid Sequence↗
PubMed1990

C1 binding by mouse IgM. The effect of abnormal glycosylation at position 402 resulting from a serine to asparagine exchange at residue 406 of the mu-chain.

We have previously shown that IgM-Asn406, a mutant IgM which has asparagine in place of the serine which is normally found at position 406, also has an abnormally glycosylated mu-chain and is defective in complement-dependent cytolysis. Here we show by analyzing cyanogen bromide fragments from normal and mutant mu-chains that the site of abnormal glycosylation is at the neighboring position, Asn402. The cytolytic defect was shown to be due to impaired C1 binding. At physiological ionic strength, the C1 binding defect was estimated to be 12-fold, which correlates well with the measured defect in cytolytic activity; also, the severity of the defect in C1 binding by the mutant protein decreases with decreasing ionic strength. Kinetic studies showed that the difference in affinities is due to a proportional difference in the association rate for C1q. By comparing IgM made in the presence and absence of deoxymannojirimycin, we show further that the defect in cytolytic activity derives mostly from the abnormal oligosaccharide.

Amino Acid Sequence↗
PubMed1990

Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

Animals↗
PubMed1988

C1 binding by murine IgM. The effect of a Pro-to-Ser exchange at residue 436 of the mu-chain.

We have examined a defect in complement activation in a mutant trinitrophenyl-binding pentameric murine monoclonal IgM which has serine replacing the proline normally found at position 436 in the protein. The mutant protein showed equivalent hapten binding but a 100-fold decreased ability to initiate complement-dependent lysis of trinitrophenyl-coupled erythrocytes at physiological ionic strength (mu = 0.15). C4b deposition mediated by the mutant protein was impaired to a similar degree. C1 bound by the mutant protein showed C1s to C1-s conversion, suggesting normal activation. When measured at reduced ionic strength (mu = 0.06), the C1 and C1q binding affinity of the mutant protein was approximately one-half that of the wild type. However, the C1 binding affinity of the mutant protein showed a greater dependence upon ionic strength such that at physiological ionic strength we estimate a 50-fold lower C1 binding affinity for the mutant molecule. Kinetic studies suggested that this difference in affinity was largely attributable to differences in association rates. In addition, a fixed proportion of the mutant molecules showed no C1 binding. We conclude that the defect in complement activation occurs at the level of C1 binding. Our data support a role for the C mu 3 domain (residues 340-440) in C1 binding by IgM.

Complement Activating Enzymes↗
PubMed1988

A thermodynamic study of the interaction between human complement components C3b or C3(H2O) and factor B in solution.

The fluid-phase interaction between factor B and an activated form of C3 (C3b or C3(H2O)) is fundamental to the formation of the alternative complement pathway C3 convertase. The present study reports on the thermodynamic parameters that govern these interactions. The extrinsic fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS) and factor B were found to act as competitive ligands in binding to C3b. Thus, complex formation between C3b or C3(H2O) and factor B could be monitored by the quenching of C3b/C3(H2O)-dependent ANS fluorescence upon the addition of B. Under physiological conditions (0.5 mM Mg2+, 37 degrees C, mu = 0.15), the Ka governing the binding of C3b to B was 2.5 X 10(6) M-1, whereas the interaction of C3(H2O) with factor B was of 5-fold lower affinity. Both reactions were endothermic, with the van't Hoff enthalpy being approximately +16.0 kcal mol-1 in each case. Thus, a large positive entropy change provides the net driving force in these interactions. Although Ka increased at higher Mg2+ concentrations, this was not an enthalpy-mediated phenomenon. Taken together, these data are consistent with hydrophobic interactions being dominant in C3b.B or C3(H2O).B complex formation. The enhancement of complex formation by Mg2+ and concomitant increase in delta S suggests that the metal ion plays a role in increasing the number of hydrophobic contacts.

Algorithms↗
PubMed1987

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus).

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.

Amino Acid Sequence↗
PubMed1987

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact.

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.

Chemical Phenomena↗
PubMed1987

Biosynthesis of the third component of complement by the human monocyte-like cell line, U-937.

The human monocyte-like cell line, U-937, was shown to synthesize and secrete C3 by hemolytic assays, radioimmunoassays and metabolic labeling experiments. The daily synthesis of antigenic C3 by unstimulated U-937 cells was low (about 3 ng/10(6) cells/24 hr) over a 3 day period. Induction of the cells to differentiate into macrophage-like cells with phorbol myristate acetate (PMA), resulted in 5-fold augmentation of C3 synthesis and secretion into the culture medium. Using a plaque assay for enumerating C3 production by single cells, approx. 5% of unstimulated U-937 cells were found to secrete hemolytically active C3. The proportion of C3-plaque forming cells was increased about 6-fold in PMA stimulated cells. The synthesis of C3 by U-937 cells was reversibly inhibited by cycloheximide. Data from SDS-PAGE analyses showed that U-937 cells synthesized C3 as a precursor polypeptide chain and was capable of processing this pro-molecule into the secreted two chain form. C3 antigen immunoprecipitated from stimulated U-937 cell lysates showed an increased amount of low mol. wt material as compared to C3 antigen immunoprecipitated from the lysates of unstimulated cells. This may be attributable to increased intracellular proteolytic activity in the PMA stimulated cells. The studies show that the U-937 cell line provides a useful model for studies on the synthesis and processing of complement proteins and the physiological regulation of complement production.

Cell Line↗
PubMed1987

Biological properties of chimeric antibodies. Interaction with complement.

Genetic engineering allows the production of chimeric immunoglobulins where the same variable region is expressed in conjunction with constant regions of different species. We have compared the capacity of mouse and chimeric (mouse variable region/human constant region) immunoglobulin M (IgM) to trigger complement-dependent lysis and to bind complement component C1. Guinea pig and human C1 were bound more efficiently by mouse IgM than by chimeric IgM, whereas rat and rabbit C1 were bound more efficiently by chimeric IgM. Comparable results were obtained for complement as measured by the lysis of sensitized erythrocytes. These results indicated that differences between the human and mouse mu heavy chain constant regions define structures that are important in the C1-IgM interaction. Furthermore, species-specific differences in C1 also influence this interaction.

Animals↗
PubMed1986

Covalent binding properties of the C4A and C4B isotypes of the fourth component of human complement on several C1-bearing cell surfaces.

In a previous study we demonstrated that the thioester-mediated transacylation of the human C4B isotype onto sheep erythrocytes (ES) was approximately fourfold more efficient than that of C4A. Moreover, although C4B formed predominantly ester linkages, C4A displayed a preference for amide bond formation. We therefore suggested that the relative functional activity observed for the two isotypes would be a combined reflection of their nucleophilic preference and the surface composition of the C1-bearing target. The present study tests this hypothesis. Chemical modification of amino groups on Es with ethylacetimidate produced a twofold decrease in the C1-dependent binding of C4A isotype, while having a negligible effect on C4B binding. Furthermore, with human erythrocytes and two human leukocyte cell lines, K562 and U937, the C4B to C4A deposition ratio decreased from greater than 4 with ES to between 1.5 and 2. Irrespective of the target, C4A and C4B maintained their preference for forming amide and ester bonds, respectively. Interestingly, SDS-PAGE profiles of radiolabeled C4A and C4B, which had been covalently deposited on the various cells, suggested a further degree of transacylation specificity, as the two isotypic alpha-chains sometimes bound to different membrane components. These differences were not easily accounted for by simple differences in the abundance of the preferred nucleophile for each isotype on a given surface constituent, nor were they due to the preferential binding of one isotype to the sensitizing antibody. We speculate that nascent C4B may contain a substrate binding site that facilitates productive attack on the thioester carbonyl by molecules containing the class of nucleophile preferred by each isotype.

Acylation↗
PubMed1986

A reexamination of the role of magnesium in the human alternative pathway of complement.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

Centrifugation, Density Gradient↗
PubMed1984

Structural basis for the preferential association of autologous immunoglobulin subunits: role of the J region of the light chain.

We have used a series of sequenced V kappa 21 L-chains produced from murine myelomas to determine whether the V or J segment of the V region is responsible for dictating preferential recombination. In each competitive recombination, equimolar amounts of two different L-chains, selected on the basis of common V or J segments, were allowed to compete for a limiting amount of H-chain. It was found that the J segment of the L-chain was primarily responsible for dictating the ability of a chain to compete and that the nature of residue 96, the first residue of the J segment, was particularly important. Specifically, charged residues caused the L-chain to compete poorly against L-chains with hydrophobic side chains at this position. Furthermore, if Phe or, to a lesser extent, Tyr were present at position 96, the L-chain competed more successfully than chains with Trp-96 or Leu-96. This suggests that both the aromaticity and size of this residue were important factors in determining preferential recombination. It was also found that all other residues in VL were secondary to residue 96 in contributing to the ability of a chain to compete. Finally, unlike all previous studies, we observed a substantial number (64%) of preferred heterologous recombinations. These results are consistent with the hypothesis that the VL and VH gene rearrangements occur independently, thus resulting in random pairing of VH and VL domains.

Amino Acid Sequence↗
PubMed1984

The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4.

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.

Acylation↗
PubMed1983

Conformational changes accompanying proteolytic cleavage of human complement protein C3b by the regulatory enzyme factor I and its cofactor H. Spectroscopic and enzymological studies.

The conformational basis for the loss of C3b functional site expression following its conversion to iC3b by the regulatory proteins factor H and factor I has been examined by a number of spectroscopic techniques including the fluorescence of the extrinsic probe 8-anilino-1-naphthalenesulfonate, circular dichroism, and UV absorption difference spectroscopy. These techniques all detected significant conformational alterations accompanying the proteolytic cleavage event. To a substantial extent, the observed spectral changes tended to reverse those seen in the original conversion of native C3 to its functionally active C3b form. Similar changes were also seen when the thioester-cleaved, but peptide chain-intact, form of C3 was acted upon by the regulatory proteins. The most dramatic effect was seen in the 8-anilino-1-naphthalenesulfonate fluorescence system where the fluorescence enhancement observed upon conversion of C3 to C3b, which presumably reflects an increase in surface hydrophobicity, is lost upon further cleavage of the molecule to iC3b. This marked signal change forms the basis of a simple spectrofluorometric assay with which to study the enzymatic properties of factor I for its true substrate: the complex formed between C3b and factor H. Using this assay, factor I was found to display very tight binding of its substance, the Km at 25 degrees C being on the order of 0.025-0.1 microM, but it was also found to show a low rate of cleavage, the turnover number being less than 10 min-1 at this temperature.

Circular Dichroism↗
PubMed1983

The role of the thioester bond in C3 and C4 in the determination of the conformational and functional states of the molecule.

The numerous ligand binding properties expressed by the activated forms of C3 (C3b) and C4 (C4b) are best explained as arising from proteolytic-cleavage-induced conformational changes. The studies described above provide direct physical evidence for such conformational transitions in both complement proteins. Significantly, however, a virtually identical conformational end state was also produced by direct nucleophilic scission of the internal thioester bond in C3 and C4 in the absence of any proteolysis. Clearly, it is the integrity of this thiolactone structure that is the determining factor in maintaining the native conformation in C3 and C4. Recent studies suggest a similar conformational role for the thioester in alpha 2-macroglobulin. Proteolytic activation in all three thioester-containing molecules renders this structure more susceptible to nucleophilic or solvolytic attack. Whether this effect is mediated by a peptide-cleavage-induced increase in the electrophilicity of the reactive carbonyl, or simply by increasing the accessibility of the solvent to this structure, is as yet unknown. In the case of C3 and C4, removal of the activation peptide also has a profound effect on the kinetics of the conformational change initiated by the loss of the thioester. This kinetic constraint has made it possible to correlate the acquisition of ligand binding properties with the spectroscopically detectable conformational changes.

Circular Dichroism↗
PubMed1983

Effect on the human complement system of the major non-steroidal anti-inflammatory drugs: aspirin, indomethacin, phenylbutazone, oxyphenbutazone and sulindac.

The possibility that the major non-steroidal anti-inflammatory drugs may inhibit the complement system and thus ameliorate the acute pathological changes induced by immune complexes was investigated. Treatment of fresh human serum with indomethacin (IDM), sulindac (Su), phenylbutazone (Ph) and oxyphenbutazone (OPh) inhibited both the classical and alternative complement (C) pathway activities in a dose-dependent fashion with a 50% inhibition dose of 4.65, 1.0, 1.65 and 1.3 mg/ml respectively. Aspirin, on the other hand, had a comparatively weak anti-complementary activity. Su, Ph and OPh were shown to form complexes with C5, thereby inhibiting the interaction between C3b and C5 and the cleavage of the latter into phlogistic fragments.

Anti-Inflammatory Agents↗