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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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The sequence of the light chain genes encoding xenoantibodies in non-human primates that are most similar to the human germline gene HSIGKV134. (A.) The nucleotide sequence of genes encoding the light chain of xenoantibodies aligned with the human germline gene HSIGKV134 and the closest non-human primate germline gene, IGKVIf, is shown. The sequences of the genes encoding xenoantibody light chains at days 0 and 21 are compared. Clone numbers are indicated in parenthesis.(B.) The amino acid sequence of genes encoding the light chain of xenoantibodies.
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Figure 3: The sequence of the light chain genes encoding xenoantibodies in non-human primates that are most similar to the human germline gene HSIGKV134. (A.) The nucleotide sequence of genes encoding the light chain of xenoantibodies aligned with the human germline gene HSIGKV134 and the closest non-human primate germline gene, IGKVIf, is shown. The sequences of the genes encoding xenoantibody light chains at days 0 and 21 are compared. Clone numbers are indicated in parenthesis.(B.) The amino acid sequence of genes encoding the light chain of xenoantibodies.

Mentions: In humans and non-human primates, a restricted group of genes encodes the heavy chain of xenoantibodies, but information on light chain genes is very limited. Due to insufficient sequence information, the determination as to whether a similar restriction in light chain gene usage occurs cannot be made, nor can accurate molecular models of xenoantibodies be generated. We have previously shown that xenoantibodies encoded by the IGHV3-11 IgVH germline progenitor can bind to the gal carbohydrate when paired with the DPK9 light chain gene [6]. In non-human primates, the light chain genes encoding xenoantibodies have not been defined. We therefore prepared cDNA libraries from peripheral blood samples of non-human primates mounting active xenoantibody responses following exposure to porcine hepatocytes or heart xenografts. Our earlier studies showed that the IGHV3-11 germline progenitor encodes xenoantibodies in these non-human primates [9,10], and the same samples were now used to identify the light chain genes expressed at this time. Serum samples were initially used to verify that an induced xenoantibody response could be documented by ELISA and cells were used to produce a series of cDNA libraries [10]. We prepared 6 cDNA libraries from rhesus monkeys prior to and at day 21 following exposure to pig cells to identify the germline progenitors encoding light chains. The relative frequency of Ig gene usage by individual germline progenitors was determined by sequencing 40 random clones from each library. The results indicate that xenoantibodies expressed at day 21 were encoded by a restricted number of germline progenitors. The closest matching human germline genes were DPK5 and HSIGKV134 in the VK I family (Figures 2 and 3 respectively). The closest matching germline sequences in the rhesus monkey were IgKV1f and IgKV1z which showed 95% and 93% nucleic acid sequence homology with the day 21 post-transplant rhesus monkey genes encoding xenoantibodies [28]. To date, little information is available on VH and VL germline genes in non-human primates. The rhesus germline light chain genes are members of the V kappa 1 subgroup and are of the same canonical structural class as human light chain genes encoding single chain antibodies that bind to the gal carbohydrate. The same germline progenitors were used to encode light chains in xenoantibodies induced following immunization with porcine hepatocytes or transplantation with porcine heart xenografts. This information was used in the computer-simulated model to complete our analysis of the binding site configuration in xenoantibodies selected for expansion in primates.


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)

The sequence of the light chain genes encoding xenoantibodies in non-human primates that are most similar to the human germline gene HSIGKV134. (A.) The nucleotide sequence of genes encoding the light chain of xenoantibodies aligned with the human germline gene HSIGKV134 and the closest non-human primate germline gene, IGKVIf, is shown. The sequences of the genes encoding xenoantibody light chains at days 0 and 21 are compared. Clone numbers are indicated in parenthesis.(B.) The amino acid sequence of genes encoding the light chain of xenoantibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 3: The sequence of the light chain genes encoding xenoantibodies in non-human primates that are most similar to the human germline gene HSIGKV134. (A.) The nucleotide sequence of genes encoding the light chain of xenoantibodies aligned with the human germline gene HSIGKV134 and the closest non-human primate germline gene, IGKVIf, is shown. The sequences of the genes encoding xenoantibody light chains at days 0 and 21 are compared. Clone numbers are indicated in parenthesis.(B.) The amino acid sequence of genes encoding the light chain of xenoantibodies.
Mentions: In humans and non-human primates, a restricted group of genes encodes the heavy chain of xenoantibodies, but information on light chain genes is very limited. Due to insufficient sequence information, the determination as to whether a similar restriction in light chain gene usage occurs cannot be made, nor can accurate molecular models of xenoantibodies be generated. We have previously shown that xenoantibodies encoded by the IGHV3-11 IgVH germline progenitor can bind to the gal carbohydrate when paired with the DPK9 light chain gene [6]. In non-human primates, the light chain genes encoding xenoantibodies have not been defined. We therefore prepared cDNA libraries from peripheral blood samples of non-human primates mounting active xenoantibody responses following exposure to porcine hepatocytes or heart xenografts. Our earlier studies showed that the IGHV3-11 germline progenitor encodes xenoantibodies in these non-human primates [9,10], and the same samples were now used to identify the light chain genes expressed at this time. Serum samples were initially used to verify that an induced xenoantibody response could be documented by ELISA and cells were used to produce a series of cDNA libraries [10]. We prepared 6 cDNA libraries from rhesus monkeys prior to and at day 21 following exposure to pig cells to identify the germline progenitors encoding light chains. The relative frequency of Ig gene usage by individual germline progenitors was determined by sequencing 40 random clones from each library. The results indicate that xenoantibodies expressed at day 21 were encoded by a restricted number of germline progenitors. The closest matching human germline genes were DPK5 and HSIGKV134 in the VK I family (Figures 2 and 3 respectively). The closest matching germline sequences in the rhesus monkey were IgKV1f and IgKV1z which showed 95% and 93% nucleic acid sequence homology with the day 21 post-transplant rhesus monkey genes encoding xenoantibodies [28]. To date, little information is available on VH and VL germline genes in non-human primates. The rhesus germline light chain genes are members of the V kappa 1 subgroup and are of the same canonical structural class as human light chain genes encoding single chain antibodies that bind to the gal carbohydrate. The same germline progenitors were used to encode light chains in xenoantibodies induced following immunization with porcine hepatocytes or transplantation with porcine heart xenografts. This information was used in the computer-simulated model to complete our analysis of the binding site configuration in xenoantibodies selected for expansion in primates.

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