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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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Single chain xenoantibodies were produced using the pHEN vector and expressed as soluble antibodies.  The xenoantibodies with site-directed mutations were compared with the germline progenitor for the ability to inhibit natural antibody binding. (A.) The IGHV3-11 gene encoding xenoantibodies in human patients was cloned into the vector pHEN2.  Single chain xenoantibodies were expressed as phagemid or soluble antibodies for functional studies. (B.) The soluble antibodies were run on an SDS page gel to confirm that antibody was produced. (C.) The ability of the soluble single chain antibodies with specific site-directed mutations to block human xenoantibody binding was compared by inhibition ELISA. The ability to bind more efficiently to purified gal carbohydrate, and thereby block human xenoantibody binding more effectively was compared in this assay.  Single chain antibodies with a mutation at site 50 block the binding of human xenoantibodies most effectively.
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Figure 6: Single chain xenoantibodies were produced using the pHEN vector and expressed as soluble antibodies. The xenoantibodies with site-directed mutations were compared with the germline progenitor for the ability to inhibit natural antibody binding. (A.) The IGHV3-11 gene encoding xenoantibodies in human patients was cloned into the vector pHEN2. Single chain xenoantibodies were expressed as phagemid or soluble antibodies for functional studies. (B.) The soluble antibodies were run on an SDS page gel to confirm that antibody was produced. (C.) The ability of the soluble single chain antibodies with specific site-directed mutations to block human xenoantibody binding was compared by inhibition ELISA. The ability to bind more efficiently to purified gal carbohydrate, and thereby block human xenoantibody binding more effectively was compared in this assay. Single chain antibodies with a mutation at site 50 block the binding of human xenoantibodies most effectively.

Mentions: The CDR1 region, identified as important for xenoantibody/gal carbohydrate interaction in the molecular model, is identical in xenoantibodies induced in patients and non-human primates [5-8]. Interestingly, the CDR1 sequence of the anti-gal monoclonal antibody 22.121 that identifies the residual gal carbohydrate encoded by the iGb3S gene following knockout of the galactosyltransferase gene also utilizes this CDR1 region [7]. We selected this site for site-directed mutagenesis to create a mutation in position 31 (aspartic acid) in the xenoantibody encoded by IGHV3-11 to address whether the affinity of antigen/antibody interaction would be altered by this modification. The replacement of an aspartic acid with a serine residue was accomplished by overlap extension PCR. The gene was sequenced to confirm that the correct mutation was introduced in the desired location. Site-specific mutations were also introduced at sites 50 and 54 within the CDR2. Site 50 is frequently mutated in IgG xenoantibodies expressed in patients and in non-human primates (Figure 5, [6,9,10]). The ability of the mutated antibodies to bind to gal and block human natural antibody binding to purified gal pentasaccharide was measured using an ELISA assay. The results show that the introduction of a mutation in position 50 improved the ability of the single chain antibodies to block human natural antibody binding to purified gal, whereas mutations at other sites were less effective (Table 1, Figure 6).


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)

Single chain xenoantibodies were produced using the pHEN vector and expressed as soluble antibodies.  The xenoantibodies with site-directed mutations were compared with the germline progenitor for the ability to inhibit natural antibody binding. (A.) The IGHV3-11 gene encoding xenoantibodies in human patients was cloned into the vector pHEN2.  Single chain xenoantibodies were expressed as phagemid or soluble antibodies for functional studies. (B.) The soluble antibodies were run on an SDS page gel to confirm that antibody was produced. (C.) The ability of the soluble single chain antibodies with specific site-directed mutations to block human xenoantibody binding was compared by inhibition ELISA. The ability to bind more efficiently to purified gal carbohydrate, and thereby block human xenoantibody binding more effectively was compared in this assay.  Single chain antibodies with a mutation at site 50 block the binding of human xenoantibodies most effectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 6: Single chain xenoantibodies were produced using the pHEN vector and expressed as soluble antibodies. The xenoantibodies with site-directed mutations were compared with the germline progenitor for the ability to inhibit natural antibody binding. (A.) The IGHV3-11 gene encoding xenoantibodies in human patients was cloned into the vector pHEN2. Single chain xenoantibodies were expressed as phagemid or soluble antibodies for functional studies. (B.) The soluble antibodies were run on an SDS page gel to confirm that antibody was produced. (C.) The ability of the soluble single chain antibodies with specific site-directed mutations to block human xenoantibody binding was compared by inhibition ELISA. The ability to bind more efficiently to purified gal carbohydrate, and thereby block human xenoantibody binding more effectively was compared in this assay. Single chain antibodies with a mutation at site 50 block the binding of human xenoantibodies most effectively.
Mentions: The CDR1 region, identified as important for xenoantibody/gal carbohydrate interaction in the molecular model, is identical in xenoantibodies induced in patients and non-human primates [5-8]. Interestingly, the CDR1 sequence of the anti-gal monoclonal antibody 22.121 that identifies the residual gal carbohydrate encoded by the iGb3S gene following knockout of the galactosyltransferase gene also utilizes this CDR1 region [7]. We selected this site for site-directed mutagenesis to create a mutation in position 31 (aspartic acid) in the xenoantibody encoded by IGHV3-11 to address whether the affinity of antigen/antibody interaction would be altered by this modification. The replacement of an aspartic acid with a serine residue was accomplished by overlap extension PCR. The gene was sequenced to confirm that the correct mutation was introduced in the desired location. Site-specific mutations were also introduced at sites 50 and 54 within the CDR2. Site 50 is frequently mutated in IgG xenoantibodies expressed in patients and in non-human primates (Figure 5, [6,9,10]). The ability of the mutated antibodies to bind to gal and block human natural antibody binding to purified gal pentasaccharide was measured using an ELISA assay. The results show that the introduction of a mutation in position 50 improved the ability of the single chain antibodies to block human natural antibody binding to purified gal, whereas mutations at other sites were less effective (Table 1, Figure 6).

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