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Research Article

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

Matthias J. Feige, Melissa A. Gräwert, Moritz Marcinowski, Janosch Hennig, Julia Behnke, David Ausländer, Eva M. Herold, Jirka Peschek, Caitlin D. Castro, Martin Flajnik, Linda M. Hendershot, Michael Sattler, Michael Groll, and Johannes Buchner
  1. aDepartment of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
  2. bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
  3. cInstitute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; and
  4. dDepartment of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201

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PNAS first published May 15, 2014; https://doi.org/10.1073/pnas.1321502111
Matthias J. Feige
aDepartment of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
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  • For correspondence: matthias.feige@stjude.org
Melissa A. Gräwert
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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Moritz Marcinowski
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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Janosch Hennig
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
cInstitute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; and
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Julia Behnke
aDepartment of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
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David Ausländer
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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Eva M. Herold
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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Jirka Peschek
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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Caitlin D. Castro
dDepartment of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
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Martin Flajnik
dDepartment of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
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Linda M. Hendershot
aDepartment of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
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Michael Sattler
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
cInstitute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; and
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Michael Groll
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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Johannes Buchner
bCenter for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany;
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  1. Edited by Alan R Fersht, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved April 18, 2014 (received for review November 15, 2013)

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Significance

Sharks are among the evolutionary oldest living organisms with an immune system that possesses a number of elements similar to ours, including antibodies. In this article, we present structural insights into one of the most ancient antibodies, shedding light on the molecular evolution of the immune system and the structural features of heavy chain-only antibodies. Sharks enrich urea in their blood to prevent osmotic loss of water in the marine environment. Urea, however, denatures proteins if they are not sufficiently stable. Indeed, we find that shark antibodies are particularly stable. We pinpointed specific features responsible for their high stability and found that transplanting them into a human antibody increased its secretion.

Abstract

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.

  • protein evolution
  • antibody structure
  • protein folding
  • protein engineering

Footnotes

  • ↵1M.J.F., M.A.G., M.M., and J.H. contributed equally to this work.

  • ↵2To whom correspondence should be addressed. E-mail: matthias.feige{at}stjude.org.
  • ↵3Present address: European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, 22607 Hamburg, Germany.

  • ↵4Present address: Eidgenössische Technische Hochschule Zürich, Department of Biosystems Science and Engineering, 4058 Basel, Switzerland.

  • ↵5Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.

  • Author contributions: M.J.F., M.A.G., M.M., J.H., M.F., L.M.H., M.S., M.G., and J. Buchner designed research; M.J.F., M.A.G., M.M., J.H., J. Behnke, D.A., E.M.H., J.P., C.D.C., and M.F. performed research; M.J.F., M.A.G., M.M., J.H., J. Behnke, D.A., E.M.H., J.P., C.D.C., M.F., L.M.H., M.S., M.G., and J. Buchner analyzed data; and M.J.F., M.A.G., M.M., J.H., J. Behnke, D.A., E.M.H., J.P., C.D.C., M.F., L.M.H., M.S., M.G., and J. Buchner wrote the paper.

  • Conflict of interest statement: A patent for optimized antibodies based on the results presented in this study has been filed.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 4Q97 (C1), 4Q9B (C2), 4Q9C (C3), and 2MKL (C4)]. The NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession no. 19783).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1321502111/-/DCSupplemental.

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Structural analysis of shark antibodies
Matthias J. Feige, Melissa A. Gräwert, Moritz Marcinowski, Janosch Hennig, Julia Behnke, David Ausländer, Eva M. Herold, Jirka Peschek, Caitlin D. Castro, Martin Flajnik, Linda M. Hendershot, Michael Sattler, Michael Groll, Johannes Buchner
Proceedings of the National Academy of Sciences May 2014, 201321502; DOI: 10.1073/pnas.1321502111

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Structural analysis of shark antibodies
Matthias J. Feige, Melissa A. Gräwert, Moritz Marcinowski, Janosch Hennig, Julia Behnke, David Ausländer, Eva M. Herold, Jirka Peschek, Caitlin D. Castro, Martin Flajnik, Linda M. Hendershot, Michael Sattler, Michael Groll, Johannes Buchner
Proceedings of the National Academy of Sciences May 2014, 201321502; DOI: 10.1073/pnas.1321502111
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