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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.

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.

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Nucleotide sequences of the VH genes encoding xenoantibodies in the VH4 family. The IGVH4-59 germline progenitor encodes a proportion of xenoantibodies in patients mounting active xenoantibody responses. Shown are the sequences of immunoglobulin genes encoding xenoantibodies in patients at day 10 after placement on a bioartificial liver containing porcine hepatocytes. (A.) The nucleic acid sequence of genes encoding xenoantibodies in the VH4 family are most closely related to the human VH4-59 germline gene.Stars indicate identities in nucleic acid sequence. Clone numbers are indicated in parenthesis. Nomenclature of the germline genes, alignments and numbering are based on the human immunoglobulin germline gene table [68]. (B.) Translated amino acid sequences of VH4 genes encoding xenoantibodies compared with the amino acid sequence of the closest human germline progenitor VH4-59.
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Figure 1: Nucleotide sequences of the VH genes encoding xenoantibodies in the VH4 family. The IGVH4-59 germline progenitor encodes a proportion of xenoantibodies in patients mounting active xenoantibody responses. Shown are the sequences of immunoglobulin genes encoding xenoantibodies in patients at day 10 after placement on a bioartificial liver containing porcine hepatocytes. (A.) The nucleic acid sequence of genes encoding xenoantibodies in the VH4 family are most closely related to the human VH4-59 germline gene.Stars indicate identities in nucleic acid sequence. Clone numbers are indicated in parenthesis. Nomenclature of the germline genes, alignments and numbering are based on the human immunoglobulin germline gene table [68]. (B.) Translated amino acid sequences of VH4 genes encoding xenoantibodies compared with the amino acid sequence of the closest human germline progenitor VH4-59.

Mentions: We have used an anchor-ELISA PCR analysis to demonstrate that xenoantibodies are predominantly encoded by genes in the in the VH3 family and a smaller proportion of these antibodies are encoded by genes in the VH4 family [6,10]. The production of cDNA libraries from the peripheral blood of patients mounting active xenograft responses has allowed us to apply colony filter hybridization and nucleic acid sequencing to identify the germline progenitors encoding these antibodies. We identified the IGHV3-11 and IGHV3-74 germline genes as the progenitors of xenoantibodies produced by the VH3 family of immunoglobulin genes, however the progenitors encoding xenoantibodies in the VH4 family were not previously identified. To complete the structural analysis of immunoglobulin genes encoding xenoantibodies, we produced five VH4 cDNA libraries from peripheral blood samples obtained from patients mounting active xenoantibody responses to pig cells. The cDNA libraries were used to determine whether an increase in gene expression associated with any of the germline progenitors in the VH4 family could be identified when comparing Ig gene usage in patients prior to and following exposure to pig cells. On the basis of immunoglobulin gene sequencing of 48 genes in each library, the relative expression of the VH4-59 germline gene was increased from 5–8% at day 0 to 43–44% at day 10 post-xenoantigen exposure. The nucleotide and animo acid sequence of six of these genes is shown in Figure 1. The genes encoding IgM xenoantibodies in the VH4 family, similar to those encoding VH3 xenoantibodies, are expressed in germline configuration. We have previously reported that xenoantibodies encoded by the VH3 family are structurally-related and can be placed in one of seven structural classes of immunoglobulin genes [6]. Xenoantibodies in the VH4 family are characterized by a 1-1 canonical structure. This data indicates that the shape of the binding pocket of this small group of antibodies differs from that encoded by the IGHV3-11 germline genes.


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)

Nucleotide sequences of the VH genes encoding xenoantibodies in the VH4 family. The IGVH4-59 germline progenitor encodes a proportion of xenoantibodies in patients mounting active xenoantibody responses. Shown are the sequences of immunoglobulin genes encoding xenoantibodies in patients at day 10 after placement on a bioartificial liver containing porcine hepatocytes. (A.) The nucleic acid sequence of genes encoding xenoantibodies in the VH4 family are most closely related to the human VH4-59 germline gene.Stars indicate identities in nucleic acid sequence. Clone numbers are indicated in parenthesis. Nomenclature of the germline genes, alignments and numbering are based on the human immunoglobulin germline gene table [68]. (B.) Translated amino acid sequences of VH4 genes encoding xenoantibodies compared with the amino acid sequence of the closest human germline progenitor VH4-59.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Nucleotide sequences of the VH genes encoding xenoantibodies in the VH4 family. The IGVH4-59 germline progenitor encodes a proportion of xenoantibodies in patients mounting active xenoantibody responses. Shown are the sequences of immunoglobulin genes encoding xenoantibodies in patients at day 10 after placement on a bioartificial liver containing porcine hepatocytes. (A.) The nucleic acid sequence of genes encoding xenoantibodies in the VH4 family are most closely related to the human VH4-59 germline gene.Stars indicate identities in nucleic acid sequence. Clone numbers are indicated in parenthesis. Nomenclature of the germline genes, alignments and numbering are based on the human immunoglobulin germline gene table [68]. (B.) Translated amino acid sequences of VH4 genes encoding xenoantibodies compared with the amino acid sequence of the closest human germline progenitor VH4-59.
Mentions: We have used an anchor-ELISA PCR analysis to demonstrate that xenoantibodies are predominantly encoded by genes in the in the VH3 family and a smaller proportion of these antibodies are encoded by genes in the VH4 family [6,10]. The production of cDNA libraries from the peripheral blood of patients mounting active xenograft responses has allowed us to apply colony filter hybridization and nucleic acid sequencing to identify the germline progenitors encoding these antibodies. We identified the IGHV3-11 and IGHV3-74 germline genes as the progenitors of xenoantibodies produced by the VH3 family of immunoglobulin genes, however the progenitors encoding xenoantibodies in the VH4 family were not previously identified. To complete the structural analysis of immunoglobulin genes encoding xenoantibodies, we produced five VH4 cDNA libraries from peripheral blood samples obtained from patients mounting active xenoantibody responses to pig cells. The cDNA libraries were used to determine whether an increase in gene expression associated with any of the germline progenitors in the VH4 family could be identified when comparing Ig gene usage in patients prior to and following exposure to pig cells. On the basis of immunoglobulin gene sequencing of 48 genes in each library, the relative expression of the VH4-59 germline gene was increased from 5–8% at day 0 to 43–44% at day 10 post-xenoantigen exposure. The nucleotide and animo acid sequence of six of these genes is shown in Figure 1. The genes encoding IgM xenoantibodies in the VH4 family, similar to those encoding VH3 xenoantibodies, are expressed in germline configuration. We have previously reported that xenoantibodies encoded by the VH3 family are structurally-related and can be placed in one of seven structural classes of immunoglobulin genes [6]. Xenoantibodies in the VH4 family are characterized by a 1-1 canonical structure. This data indicates that the shape of the binding pocket of this small group of antibodies differs from that encoded by the IGHV3-11 germline genes.

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