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A molecular model for an antibody that binds Lex and sialyl Lex
278
Abstracts
different decreases in binding to NA. The H-bond formed by Tyr32 makes an important contribution to binding; loss of phenolic OH results in ~ 20-fold decrease in K, whereas the Tryl00aPhe mutant shows only a 2-fold decrease compared to the parent scFv. These results show that the residues identified from structural studies make important contributions to the binding interactions and that single residue changes can have a profound effect on the interaction. Defective secretion of VH CDR2 mutant antibodies is characterized by increased association of the mutant heavy chains with molecular chaperones and is not a property of the fight chain. T.M. Martin, G.D. Wiens, C. Kowalczyk, M.B. Rittenberg,
Dept. of Molecular Mircobiology qnd Immunology, Oregon Health Sciences University, Portland, OR 97201, USA. To understand antibody function, we are using an in vitro random mutagenesis system to mimic the negative effetcs of somatic hypermutation. This approach has revealed that VH structure affects antibody secretion. Four mutants differing from the wild type (WT) T15 antibody by 2 - 4 AA changes in the H chain CDR2 were shown to have defective secretion. To understand the structural basis for this defect, we have assessed various molecular characteristics of these mutants. We have already shown that normal levels of H chain are produced, and that some assembly with L chains can occur based on anti-idiotype recognition and, for one mutant, antigen binding. Here, pulse-chase/immunoprecipitation experiments reveal that the half life of each mutant H chain differs appreciably from the intact WT protein. WT antibody is secreted efficiently, with an intracellular half-live of ~ 3 h, while the mutant H chains have intraceUular half-lives ranging from 12 to > 26 h. Mutant H chains are associated with the molecular chaperones, BiP and Grp94, at levels that are 5-15-fold greater than the WT H chain. These data indicate that the kinetics of assembly and transport are highly inefficient and that the mutant H chains accumulate intracellulary, most likely in the ER. The T15 L chain, Vx22, unlike most other L chains, is not secreted in the absence of H chain. To determine if this feature of Vx22 plays a role with the low secretor phenotype, the WT and mutant H chains were expressed with two other L chains, Vxl and VA1, which are secreted in the absence of H chain. These new H + L pairings were secreted efficiently when combined with the WT H chain, but not with the mutant H chains, indicating that the Vx22 L chain is not uniquely responsible for defective secretion. This study demonstrates that mutations in a H chain hypervariable region can disrupt immunoglobulin secretion, in contrast to the paradigm that CDR loops do not make significant structural contributions to immunoglobulin functions other than antigen binding. NIH support: A1 14985, AI 26827 and EY 07123. Comparison of the N10 structural, functional and energetic epitodes of staphylococcal nuclense. Samuel S. Perdue, David C, Benjamin, Department of Microbi-
ology and the Beirne B. Carter Centerfor Immunology Research, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
We have used structural, functional and computational analysis to investigate the interaction between the model antigen staphylococcal nuclease (SNase) and the monoclonal antibody N10. The 'structural' epitope determined from the X-ray crystal structure of the complex is comprised of 17 contact residues. By calculating the 'energetic' epitope from the minimized crystal coordinates, we predicted an epitope consisting of seven of these residues, most of which form electrostatic contacts with N10. This epitope is dominated by two residues (Lys-70 and Glu-135) that form salt links with the antibody. Functional analysis of alanine substitution mutants revealed a very different 'functional' epitope, consisting of only four residues (two shared with the energetic epitope) and not including Lys-70 and Glu-135. Finally, we used computer modelling to construct molecular models of each alanine substitution mutant, and again calculated the energetic epitope, this time using the model coordinates. This last procedure yielded an energetic epitope which was a five-residue subset of the energetic epitope determined from the minimized coordinates, and again was dominated by Lys-70 and Glu-135. We report here an in depth comparison of the four epitopes, and discuss the importance of electrostatic interactions and solvent accessibility to the formation of this complex formation. We also discuss the limitiations of alanine scanning mutagenesis in accurate determination of the true energetic epitope. A molecular model for an antibody that hinds Le x and sialyl Le x. Jianxia Qiu a, Quang Dinh ~, Nanping Weng ~, Donal M. Marcus a, Eduardo A. Padlan b, aBaylor Col. of Med., Houston, TX
77030, bNIDDK, NIH, Bethesda, MD 20892, USA. We previously studied a panel of monoclonal antibodies (mAbs) that bind to the Le x (3-FL, CD15) carbohydrate epitope. All of the Abs are IgM, K encoded by VH441 and VK24B and exhibited low affinity to Ag. A high affinity mAb, clone 24, was obtained from a phage display library and selected by a LeX-BSA conjugate. This Ab binds equally well to Le x and sialyl Le x, and it is encoded by VH441 and VK Ox-1 family genes; clone 24 binds Le X 100 x more strongly than PMS1, which is one of the original mAbs. A computer model was constructed for the Fv fragment of clone 24, based on sequence similarity with Abs of known 3-D structures. In the modeling of the VH domain of clone 24, the V H of J539(PDB2FBJ) was used as template, except for CDR3H which was based on the corresponding segment in D1.3(PDB 1VFB). For the VL domain of lone 24, the VL of ANO2(PDB 1BAF) was used as template, except for CDR1-Land CDR3-L which were based on the corresponding segemnts in 1F7(PDB 1FIG) and 26-10(PDB IIGJ), respectively. The quaternary structure of 1F7 Fv was assumed for clone 24. The model was built using the molecular graphics program FRODO and energy-minimized using X-PLOR. The CDR surface of the clone 24 model shows a large cleft that resembles the model of PM81, despite their difference in specificity and affinity. The model of clone 24 differs from the structure of anti-galactan Ab J539 and the model of another anti-carbohydrate mAb 3A9, despite the fact that the latter two Abs are also encoded by VH441 and VK Ox-1 genes. J539 and 3A9 display shallow pockets on their surfaces.
Abstracts
different decreases in binding to NA. The H-bond formed by Tyr32 makes an important contribution to binding; loss of phenolic OH results in ~ 20-fold decrease in K, whereas the Tryl00aPhe mutant shows only a 2-fold decrease compared to the parent scFv. These results show that the residues identified from structural studies make important contributions to the binding interactions and that single residue changes can have a profound effect on the interaction. Defective secretion of VH CDR2 mutant antibodies is characterized by increased association of the mutant heavy chains with molecular chaperones and is not a property of the fight chain. T.M. Martin, G.D. Wiens, C. Kowalczyk, M.B. Rittenberg,
Dept. of Molecular Mircobiology qnd Immunology, Oregon Health Sciences University, Portland, OR 97201, USA. To understand antibody function, we are using an in vitro random mutagenesis system to mimic the negative effetcs of somatic hypermutation. This approach has revealed that VH structure affects antibody secretion. Four mutants differing from the wild type (WT) T15 antibody by 2 - 4 AA changes in the H chain CDR2 were shown to have defective secretion. To understand the structural basis for this defect, we have assessed various molecular characteristics of these mutants. We have already shown that normal levels of H chain are produced, and that some assembly with L chains can occur based on anti-idiotype recognition and, for one mutant, antigen binding. Here, pulse-chase/immunoprecipitation experiments reveal that the half life of each mutant H chain differs appreciably from the intact WT protein. WT antibody is secreted efficiently, with an intracellular half-live of ~ 3 h, while the mutant H chains have intraceUular half-lives ranging from 12 to > 26 h. Mutant H chains are associated with the molecular chaperones, BiP and Grp94, at levels that are 5-15-fold greater than the WT H chain. These data indicate that the kinetics of assembly and transport are highly inefficient and that the mutant H chains accumulate intracellulary, most likely in the ER. The T15 L chain, Vx22, unlike most other L chains, is not secreted in the absence of H chain. To determine if this feature of Vx22 plays a role with the low secretor phenotype, the WT and mutant H chains were expressed with two other L chains, Vxl and VA1, which are secreted in the absence of H chain. These new H + L pairings were secreted efficiently when combined with the WT H chain, but not with the mutant H chains, indicating that the Vx22 L chain is not uniquely responsible for defective secretion. This study demonstrates that mutations in a H chain hypervariable region can disrupt immunoglobulin secretion, in contrast to the paradigm that CDR loops do not make significant structural contributions to immunoglobulin functions other than antigen binding. NIH support: A1 14985, AI 26827 and EY 07123. Comparison of the N10 structural, functional and energetic epitodes of staphylococcal nuclense. Samuel S. Perdue, David C, Benjamin, Department of Microbi-
ology and the Beirne B. Carter Centerfor Immunology Research, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
We have used structural, functional and computational analysis to investigate the interaction between the model antigen staphylococcal nuclease (SNase) and the monoclonal antibody N10. The 'structural' epitope determined from the X-ray crystal structure of the complex is comprised of 17 contact residues. By calculating the 'energetic' epitope from the minimized crystal coordinates, we predicted an epitope consisting of seven of these residues, most of which form electrostatic contacts with N10. This epitope is dominated by two residues (Lys-70 and Glu-135) that form salt links with the antibody. Functional analysis of alanine substitution mutants revealed a very different 'functional' epitope, consisting of only four residues (two shared with the energetic epitope) and not including Lys-70 and Glu-135. Finally, we used computer modelling to construct molecular models of each alanine substitution mutant, and again calculated the energetic epitope, this time using the model coordinates. This last procedure yielded an energetic epitope which was a five-residue subset of the energetic epitope determined from the minimized coordinates, and again was dominated by Lys-70 and Glu-135. We report here an in depth comparison of the four epitopes, and discuss the importance of electrostatic interactions and solvent accessibility to the formation of this complex formation. We also discuss the limitiations of alanine scanning mutagenesis in accurate determination of the true energetic epitope. A molecular model for an antibody that hinds Le x and sialyl Le x. Jianxia Qiu a, Quang Dinh ~, Nanping Weng ~, Donal M. Marcus a, Eduardo A. Padlan b, aBaylor Col. of Med., Houston, TX
77030, bNIDDK, NIH, Bethesda, MD 20892, USA. We previously studied a panel of monoclonal antibodies (mAbs) that bind to the Le x (3-FL, CD15) carbohydrate epitope. All of the Abs are IgM, K encoded by VH441 and VK24B and exhibited low affinity to Ag. A high affinity mAb, clone 24, was obtained from a phage display library and selected by a LeX-BSA conjugate. This Ab binds equally well to Le x and sialyl Le x, and it is encoded by VH441 and VK Ox-1 family genes; clone 24 binds Le X 100 x more strongly than PMS1, which is one of the original mAbs. A computer model was constructed for the Fv fragment of clone 24, based on sequence similarity with Abs of known 3-D structures. In the modeling of the VH domain of clone 24, the V H of J539(PDB2FBJ) was used as template, except for CDR3H which was based on the corresponding segment in D1.3(PDB 1VFB). For the VL domain of lone 24, the VL of ANO2(PDB 1BAF) was used as template, except for CDR1-Land CDR3-L which were based on the corresponding segemnts in 1F7(PDB 1FIG) and 26-10(PDB IIGJ), respectively. The quaternary structure of 1F7 Fv was assumed for clone 24. The model was built using the molecular graphics program FRODO and energy-minimized using X-PLOR. The CDR surface of the clone 24 model shows a large cleft that resembles the model of PM81, despite their difference in specificity and affinity. The model of clone 24 differs from the structure of anti-galactan Ab J539 and the model of another anti-carbohydrate mAb 3A9, despite the fact that the latter two Abs are also encoded by VH441 and VK Ox-1 genes. J539 and 3A9 display shallow pockets on their surfaces.