16 results on '"David E. Isenman"'
Search Results
2. The FGL2-FcγRIIB pathway: A novel mechanism leading to immunosuppression
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Li Zhang, Elisa Leung, Justin Manuel, Wei He, Jun Diao, David R. Grant, David A. Clark, Reginald M. Gorczynski, Ming F. Liu, Melville J. Phillips, Jennifer Crookshank, David E. Isenman, Hao Liu, Gary A. Levy, Itay Shalev, Myron I. Cybulsky, and Mark S. Cattral more...
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Immunology ,Antigen-Presenting Cells ,Biology ,Flow cytometry ,Pathogenesis ,Mice ,Immune Tolerance ,medicine ,Animals ,Transplantation, Homologous ,Immunology and Allergy ,Receptor ,Mice, Inbred BALB C ,medicine.diagnostic_test ,Graft Survival ,Receptors, IgG ,Fibrinogen ,Dendritic Cells ,Skin Transplantation ,FGL2 ,Cell biology ,Mice, Inbred C57BL ,Cell culture ,Apoptosis ,biology.protein ,Fc-Gamma Receptor ,Female ,Antibody - Abstract
Fibrinogen-like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM-derived DC and T-cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2-specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FcgammaR)IIB and FcgammaRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FcgammaRIIB and FcgammaRIII, or deficiency of these receptors, abrogated FGL2 binding. FGL2 inhibited the maturation of BMDC from FcgammaRIIB+/+ mice but not from FcgammaRIIB(-/-) mice and induced apoptosis in the FcgammaRIIB+ mouse B-cell line (A20) but not the A20IIA1.6 cell line that does not express FcgammaRIIB. Recombinant FGL2 infused into FcgammaRIIB+/+ (C57BL/6J, H-2b) mice but not FcgammaRIIB(-/-) mice inhibited rejection of fully mismatched BALB/cJ (H-2d) skin allografts. The identification of specific receptor binding has important implications for the pathogenesis of immune-mediated disease and suggests a potential for targeted FGL2 therapy. more...
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- 2008
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Catalog
3. Interaction of Human Complement with Sbi, a Staphylococcal Immunoglobulin-binding Protein
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Jean M. H. van den Elsen, Elisa Leung, Stefan Bagby, Julia Burman, D. I. Svergun, Karen Atkins, Lea Lango, Maghnus O'Seaghdha, David E. Isenman, Timothy J. Foster, and Pau Bernadó
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Complement component 2 ,Cell Biology ,Plasma protein binding ,Biology ,medicine.disease_cause ,Biochemistry ,Immunoglobulin G ,Complement system ,Staphylococcus aureus ,Alternative complement pathway ,medicine ,Extracellular ,biology.protein ,Anaphylatoxin ,Molecular Biology - Abstract
Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and β2-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption. more...
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- 2008
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4. The Complement Regulator C4b-Binding Protein (C4BP) Interacts with both the C4c and C4dg Subfragments of the Parent C4b Ligand: Evidence for Synergy in C4BP Subsite Binding
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David E. Isenman, Anna M. Blom, Liliana Clemenza, and Elisa Leung
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Binding Sites ,biology ,Heparin ,Chemistry ,Stereochemistry ,Complement C4b-Binding Protein ,Protein subunit ,Binding protein ,Osmolar Concentration ,Cooperative binding ,Complement C4 ,Surface Plasmon Resonance ,Ligands ,Ligand (biochemistry) ,Biochemistry ,Peptide Fragments ,Complement system ,Mutation ,biology.protein ,Humans ,Amino Acid Sequence ,Binding site ,Binding domain ,Complement control protein - Abstract
C4b-binding protein (C4BP) is a multimeric serum protein that is a potent regulator of the classical and lectin complement pathways. The binding site for C4b has been localized to complement control protein (CCP) domains 1-3 of the C4BP alpha-chain and, in particular, to a cluster of positively charged amino acids predicted to be at the interface between CCP 1 and CCP 2. To determine the regions of C4b contributing to C4BP binding, we have examined via surface plasmon resonance technology the binding of the C4c and C4dg subfragments of C4b to C4BP. At half-physiologic ionic strength, specific and saturable binding was observed for both C4c and C4dg. C4c exhibited much greater ionic strength sensitivity in its binding than did C4dg. Analysis of the effect on binding of the subfragments to various C4b-binding-defective C4BP mutants, together with cross-competition experiments, suggests that the subsites in C4BP for C4c and C4dg are adjacent, but distinct. Additionally, we observed synergy in subsite filling such that the presence of C4dg enhanced the extent of C4c binding over its basal level, and vice versa. The enhanced binding of C4c in the presence of C4dg was not due to an increase in affinity but rather reflected a 2-3-fold increase in the number of sites capable of binding C4c. This suggests the existence of a conformational equilibrium between high- and low-affinity states in the C4c binding subsite within each C4BP subunit, an equilibrium which is shifted in favor of the high-affinity state by the filling of the C4dg subsite. more...
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- 2006
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5. Cross-Neutralizing Human Monoclonal Anti-HIV-1 Antibody 2F5: Preparation and Crystallographic Analysis of the Free and Epitope-Complexed Forms of its F ab Fragment
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Renate Kunert, Emil F. Pai, Steve Bryson, Jason Ho, Michèl R. Klein, Rosemary C. Hynes, Annie Cunningham, Hermann Katinger, Brian H. Barber, and David E. Isenman
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chemistry.chemical_classification ,Linear epitope ,biology ,Mimotope ,medicine.drug_class ,Peptide ,General Medicine ,Monoclonal antibody ,Gp41 ,Biochemistry ,Molecular biology ,Epitope ,Crystallography ,Viral envelope ,chemistry ,Structural Biology ,medicine ,biology.protein ,Antibody - Abstract
The human monoclonal antibody 2F5 is a potent neutralizer of most clades of HIV-1 and possesses protective effects against viral transmission. It recognizes the linear epitope ELDKWAS of the viral envelope protein gp41. As structural information about epitope recognition may help to develop an HIV-1 vaccine we initiated crystallographic analyses of mAb 2F5 and its epitope complex. We now report the preparation of the corresponding Fab fragments, complexation with the epitope peptide, and crystallization of free mAb 2F5 Fab as well as the peptide complex. Both crystal forms are well suited for high-resolution structural analysis. more...
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- 2001
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6. Identification of a secondary Fc gamma RI binding site within a genetically engineered human IgG antibody
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M. S. Chappel, David E. Isenman, R. Oomen, Yuan-Yuan Xu, and Michel H. Klein
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chemistry.chemical_classification ,Expression vector ,biology ,Chemistry ,Cell Biology ,Transfection ,Biochemistry ,Molecular biology ,Immunoglobulin G ,Amino acid ,biology.protein ,Binding site ,Threonine ,Antibody ,Molecular Biology ,Peptide sequence - Abstract
Although human IgG2 is not cytophilic, we have shown previously that an IgG2 antibody expressing the sequence PLLGG (underline = substitution) spanning CH2 domain residues 233-237 (Eu numbering) displayed IgG1-like Fc gamma RI binding activity. In contrast, IgG1 PLLGG exhibited 3-fold less affinity, whereas IgG2 ELLGG was 3-fold more active than native IgG1. These results suggested that additional site(s) conferred enhanced binding properties to the engineered, cytophilic IgG2 variant. These sites were shown to reside in the IgG2 CH2 domain, since the IgG1 CH2 module did not have enhanced activity in a panel of hybrid IgG1/IgG2 antibodies. To map these sites further, human IgG1 and IgG2 constant region gene segments were modified to allow reciprocal COOH-terminal half segment exchanges of CH2 exons. These were cloned into a pSV2neo expression vector bearing a rearranged MOPC 315 heavy chain variable region gene and transfected into a MOPC 315 heavy chain deletion mutant. The dinitrophenol affinity-purified IgGs were radiolabeled and assessed for Fc gamma RI binding activity in direct binding assays using U937 cells. The COOH terminus of the IgG2 CH2 domain was found to contain accessory site(s) since it enhanced the binding properties of both IgG1 PLLGG and native IgG1. In contrast, grafting of the COOH terminus of the IgG1 CH2 domain onto IgG2 PLLGG and IgG2 ELLGG diminished their cytophilic activity. The amino acid responsible for the enhancing properties of the COOH terminus of the IgG2 CH2 domain was shown to be threonine 339, since IgG1 PLLGG/Thr339 displayed increased Fc gamma RI binding affinity. Kinetics studies revealed that this is accomplished through an increase in the forward rate constant of the IgG-Fc gamma RI interaction. more...
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- 1993
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7. 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)
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David E. Isenman and A Taniguchi-Sidle
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chemistry.chemical_classification ,biology ,Binding protein ,Integrin ,CD18 ,Peptide ,Cell Biology ,Complement receptor ,Biochemistry ,Integrin alpha M ,chemistry ,parasitic diseases ,biology.protein ,iC3b ,Site-directed mutagenesis ,Molecular Biology - Abstract
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. more...
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- 1992
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8. Crystal structure of the complex between the F(ab)' fragment of the cross-neutralizing anti-HIV-1 antibody 2F5 and the F(ab) fragment of its anti-idiotypic antibody 3H6
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Renate Kunert, David E. Isenman, Emil F. Pai, Jean-Philippe Julien, Steve Bryson, and Hermann Katinger
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Idiotype ,Models, Molecular ,medicine.drug_class ,viruses ,Molecular Sequence Data ,HIV Antibodies ,Monoclonal antibody ,Gp41 ,Crystallography, X-Ray ,Epitope ,Epitopes ,Immunoglobulin Fab Fragments ,Mice ,Structural Biology ,Neutralization Tests ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Binding Sites ,biology ,Antibodies, Monoclonal ,Molecular biology ,HIV Envelope Protein gp41 ,Antibodies, Anti-Idiotypic ,biology.protein ,HIV-1 ,Paratope ,Antibody - Abstract
The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I beta-turn centered on gp41 residues (664)DKW(666); in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F(ab) and 2F5 F(ab)'. Ab2/3H6 F(ab) binds to 2F5 F(ab)' via a helix-like protrusion formed by residues (58(H))RYSPSLNTRL(67(H)) of the 2F5 F(ab)' variable domain and proximal to but not overlapping with the gp41 (664)DKW(666) epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2gamma class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 (662)ELDKWAS(668) epitope. more...
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- 2008
9. 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
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David E. Isenman, M. S. Chappel, Keith J. Dorrington, Yuan-Yuan Xu, Michel H. Klein, and Margaret Everett
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Molecular Sequence Data ,Receptors, Fc ,Biology ,Binding, Competitive ,Immunoglobulin G ,law.invention ,Cell Line ,Mice ,law ,Sequence Homology, Nucleic Acid ,Animals ,Humans ,Amino Acid Sequence ,Binding site ,Peptide sequence ,chemistry.chemical_classification ,Multidisciplinary ,Chimera ,Point mutation ,Receptors, IgG ,Molecular biology ,Antigens, Differentiation ,Recombinant Proteins ,Amino acid ,Kinetics ,Biochemistry ,chemistry ,biology.protein ,Recombinant DNA ,Mutagenesis, Site-Directed ,Fc-Gamma Receptor ,Binding Sites, Antibody ,Antibody ,Protein Multimerization ,Plasmacytoma ,Research Article - Abstract
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. more...
- Published
- 1991
10. Interaction of human complement with Sbi, a staphylococcal immunoglobulin-binding protein
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David E. Isenman, Julia Burman, Elisa Leung, and Jean M. H. van den Elsen
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biology ,Biochemistry ,Chemistry ,Immunology ,Protein A/G ,biology.protein ,Protein G ,Molecular Biology ,Immunoglobulin-binding protein ,Complement (complexity) - Published
- 2007
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11. Structure-guided identification of C3d residues essential for binding to complement receptor 2
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Liliana Clemenza and David E. Isenman
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Pharmacology ,Biochemistry ,biology ,Complement component 2 ,CD46 ,Chemistry ,Complement receptor 2 ,Lectin pathway ,Factor H ,biology.protein ,Complement component 6 ,CFHR5 ,Complement control protein - Published
- 2000
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12. A reexamination of the role of magnesium in the human alternative pathway of complement
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Edward L.G. Pryzdial and David E. Isenman
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Circular dichroism ,Complement Pathway, Alternative ,Immunology ,Complement C3-C5 Convertases ,Sulfonic acid ,Hemolysis ,Complement factor B ,Centrifugation, Density Gradient ,Humans ,Magnesium ,Complement Activation ,Molecular Biology ,chemistry.chemical_classification ,biology ,C3-convertase ,Enzyme ,Biochemistry ,chemistry ,Complement C3b ,biology.protein ,Alternative complement pathway ,Complement Factor D ,Electrophoresis, Polyacrylamide Gel ,Factor D ,Ultracentrifuge ,Complement Factor B - Abstract
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. more...
- Published
- 1986
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13. A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)
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David E. Isenman, S E Spycher, R H Painter, and Sudha Arya
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Gel electrophoresis ,animal structures ,biology ,Kunitz STI protease inhibitor ,Chemistry ,Proteolytic enzymes ,Cell Biology ,Trypsin ,Biochemistry ,alpha-2-Macroglobulin ,Protein structure ,medicine ,biology.protein ,Molecular Biology ,Polyacrylamide gel electrophoresis ,Peptide sequence ,medicine.drug - Abstract
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. more...
- Published
- 1987
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14. Folding pathways of immunoglobulin domains. The folding kinetics of the C.gamma.3 domain of human IgG1
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Doron Lancet, Israel Pecht, and David E. Isenman
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Protein Denaturation ,biology ,Protein Conformation ,Cytochrome c ,Dimer ,Kinetics ,Tryptophan ,Immunoglobulin domain ,Guanidines ,Biochemistry ,Fluorescence ,Immunoglobulin Fc Fragments ,Folding (chemistry) ,chemistry.chemical_compound ,Crystallography ,Spectrometry, Fluorescence ,chemistry ,Immunoglobulin G ,biology.protein ,Native state ,Humans ,Disulfides ,Guanidine ,Mathematics - Abstract
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. more...
- Published
- 1979
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15. Correlation between the exposure of aromatic chromophores at the surface of the Fc domains of immunoglobulin G and their ability to bind complement
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David E. Isenman, R. H. Painter, Ellerson, and Keith J. Dorrington
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Circular dichroism ,Protein Denaturation ,Stereochemistry ,Protein Conformation ,Dimer ,Biochemistry ,Immunoglobulin G ,Iodine Radioisotopes ,chemistry.chemical_compound ,Structure-Activity Relationship ,Complement C1 ,Side chain ,Lactoperoxidase ,Binding Sites ,biology ,Spectrum Analysis ,Tryptophan ,Complement System Proteins ,Chromophore ,Hydrogen-Ion Concentration ,Fragment crystallizable region ,Pepsin A ,Immunoglobulin Fc Fragments ,Monomer ,chemistry ,biology.protein ,Solvents ,Tyrosine ,Spectrophotometry, Ultraviolet - Abstract
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. more...
- Published
- 1977
16. Expression of biological effector functions by immunoglobulin G molecules lacking the hinge region
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Keith J. Dorrington, Nicole Haeffner-Cavaillon, Manuel A. Navia, David E. Isenman, Claude Rivat, David R. Davies, and Michèl R. Klein
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Multidisciplinary ,Receptors, Fc ,Biology ,Fragment crystallizable region ,Molecular biology ,Immunoglobulin G ,Complement system ,Classical complement pathway ,Mice ,Cell culture ,Complement C1 ,biology.protein ,Immunoglobulin heavy chain ,Animals ,Humans ,Receptor ,Protein A ,Immunoglobulin Constant Regions ,Immunoglobulin Heavy Chains ,Staphylococcal Protein A ,Complement Activation ,Research Article - Abstract
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. more...
- Published
- 1981
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