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Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens.

Kearns-Jonker M, Barteneva N, Mencel R, Hussain N, Shulkin I, Xu A, Yew M, Cramer DV - BMC Immunol. (2007)

Bottom Line: This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3.Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis.Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.

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Affiliation: Department of Cardiothoracic Surgery, Saban Research Institute of the Children's Hospital of Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA 90027 USA. mkearns@chla.usc.edu

ABSTRACT

Background: Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling.

Results: The VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding.

Conclusion: Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.

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Amino acid translation of the sequences of genes encoding human xenoantibodies. (A.) The amino acid translation of immunoglobulin heavy chain genes in the VH3 family encoding xenoantibodies at days 10 and 21 following exposure to porcine hepatocytes.  The sequences at day 10 are expressed in germline configuration, however, the sequences at day 21 demonstrate the onset of mutations. Site-directed mutations were introduced into the germline gene in positions shown. (B.) The amino acid sequence of the genes encoding the light chain of a human xenoantibody that binds to the gal carbohydrate is shown compared with the closest germline progenitor, DPK9.
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Figure 5: Amino acid translation of the sequences of genes encoding human xenoantibodies. (A.) The amino acid translation of immunoglobulin heavy chain genes in the VH3 family encoding xenoantibodies at days 10 and 21 following exposure to porcine hepatocytes. The sequences at day 10 are expressed in germline configuration, however, the sequences at day 21 demonstrate the onset of mutations. Site-directed mutations were introduced into the germline gene in positions shown. (B.) The amino acid sequence of the genes encoding the light chain of a human xenoantibody that binds to the gal carbohydrate is shown compared with the closest germline progenitor, DPK9.

Mentions: The structure of the binding site was addressed using computer-simulated molecular models generated by homology modeling (Figure 4). Computer-assisted homology-based modeling provides a reliable method of defining the three-dimensional structure of the binding pocket of xenoantibodies. Using this methodology, antibodies whose structure has been defined by crystallography can be used as a template for structural studies on an antibody of interest. Three-dimensional structural data obtained by modeling with homology-based techniques agrees well when compared with data determined by x-ray crystallographic techniques [32]. We used the AUTODOCK and DOCK programs for our model to identify contact sites relevant for xenoantibody binding to the gal carbohydrate (Arthur Olson, La Jolla, CA), [33,34]. Key contact sites relevant for optimal xenoantibody/gal carbohydrate interaction were identified by docking the gal carbohydrate in trisaccharide and pentasaccharide forms into the computer-simulated model of the IGHV3-11 xenoantibody. Energy scores were computed and the model selected on the basis of the lowest energy conformation. The results of the computer-assisted model indicate that the light chain, heavy chain and CDR3 region each contribute to gal saccharide binding. Key contact sites include positions 31and 33 of the CDR1, 50, 57 and 58 of the CDR2, and 99–103 of the CDR3. Positions 32 and 49 of the light chain also contribute to optimal binding to gal tri and pentasaccharide. Induced xenoantibodies and antibodies that demonstrate the ability to bind to carbohydrates have a structurally-similar binding pocket (Figure 4, [35,36]). Interestingly, mutations identified in vivo in IgG xenoantibodies expressed in patients undergoing active xenoantibody responses occur at several predicted contact sites (Table 1, Figure 5). Mutations located in positions 50, 57 and 58 in the heavy chain of IgG xenoantibodies were identified in both humans and non-human primates mounting active xenoantibody responses (Figure 5, [9,10]).


Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens.

Kearns-Jonker M, Barteneva N, Mencel R, Hussain N, Shulkin I, Xu A, Yew M, Cramer DV - BMC Immunol. (2007)

Amino acid translation of the sequences of genes encoding human xenoantibodies. (A.) The amino acid translation of immunoglobulin heavy chain genes in the VH3 family encoding xenoantibodies at days 10 and 21 following exposure to porcine hepatocytes.  The sequences at day 10 are expressed in germline configuration, however, the sequences at day 21 demonstrate the onset of mutations. Site-directed mutations were introduced into the germline gene in positions shown. (B.) The amino acid sequence of the genes encoding the light chain of a human xenoantibody that binds to the gal carbohydrate is shown compared with the closest germline progenitor, DPK9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851715&req=5

Figure 5: Amino acid translation of the sequences of genes encoding human xenoantibodies. (A.) The amino acid translation of immunoglobulin heavy chain genes in the VH3 family encoding xenoantibodies at days 10 and 21 following exposure to porcine hepatocytes. The sequences at day 10 are expressed in germline configuration, however, the sequences at day 21 demonstrate the onset of mutations. Site-directed mutations were introduced into the germline gene in positions shown. (B.) The amino acid sequence of the genes encoding the light chain of a human xenoantibody that binds to the gal carbohydrate is shown compared with the closest germline progenitor, DPK9.
Mentions: The structure of the binding site was addressed using computer-simulated molecular models generated by homology modeling (Figure 4). Computer-assisted homology-based modeling provides a reliable method of defining the three-dimensional structure of the binding pocket of xenoantibodies. Using this methodology, antibodies whose structure has been defined by crystallography can be used as a template for structural studies on an antibody of interest. Three-dimensional structural data obtained by modeling with homology-based techniques agrees well when compared with data determined by x-ray crystallographic techniques [32]. We used the AUTODOCK and DOCK programs for our model to identify contact sites relevant for xenoantibody binding to the gal carbohydrate (Arthur Olson, La Jolla, CA), [33,34]. Key contact sites relevant for optimal xenoantibody/gal carbohydrate interaction were identified by docking the gal carbohydrate in trisaccharide and pentasaccharide forms into the computer-simulated model of the IGHV3-11 xenoantibody. Energy scores were computed and the model selected on the basis of the lowest energy conformation. The results of the computer-assisted model indicate that the light chain, heavy chain and CDR3 region each contribute to gal saccharide binding. Key contact sites include positions 31and 33 of the CDR1, 50, 57 and 58 of the CDR2, and 99–103 of the CDR3. Positions 32 and 49 of the light chain also contribute to optimal binding to gal tri and pentasaccharide. Induced xenoantibodies and antibodies that demonstrate the ability to bind to carbohydrates have a structurally-similar binding pocket (Figure 4, [35,36]). Interestingly, mutations identified in vivo in IgG xenoantibodies expressed in patients undergoing active xenoantibody responses occur at several predicted contact sites (Table 1, Figure 5). Mutations located in positions 50, 57 and 58 in the heavy chain of IgG xenoantibodies were identified in both humans and non-human primates mounting active xenoantibody responses (Figure 5, [9,10]).

Bottom Line: This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3.Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis.Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cardiothoracic Surgery, Saban Research Institute of the Children's Hospital of Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA 90027 USA. mkearns@chla.usc.edu

ABSTRACT

Background: Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling.

Results: The VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding.

Conclusion: Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.

Show MeSH
Related in: MedlinePlus