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Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues

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Cell Stress and Chaperones Aims and scope

Abstract

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

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Notes

  1. According to the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature rules (Nomenclature of multiple forms of enzymes. In: Biochemical nomenclature and related documents, 2nd edition. Edited by: Liébecq C. Colchester; Portland press; 1992), we refer to differentially modified protein forms as “protein species,” as we have justified before (Benndorf and Jungblut 2015). The term isoform should be restricted to genetic variants. We refer to newly observed or rare protein species as “non-canonical protein species” which are typically found in non-canonical spots on two-dimensional gels, as opposed to the “canonical protein species” which designates the traditionally observed protein species that are found in the canonical spots on two-dimensional gels. It should also be noted that a single protein spot on two-dimensional gels may contain more than one protein species (Jungblut et al. 2008).

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  1. Rainer Benndorf and Robert R. Gilmont contributed equally to this study.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA

    Rainer Benndorf, Sahoko Hirano & Michael J. Welsh

  2. Department of Plastic and Reconstructive Surgery, University of Michigan Medical School, Ann Arbor, MI, USA

    Robert R. Gilmont

  3. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

    Richard F. Ransom

  4. Core Facility Protein Analysis, Max-Planck-Institute for Infection Biology, Berlin, Germany

    Peter R. Jungblut

  5. Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany

    Martin Bommer

  6. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    James E. Goldman

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  1. Rainer Benndorf
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Correspondence to Rainer Benndorf.

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Rats were housed in animal facilities accredited by the American Association of Laboratory Animal Care, with free access to pelleted food and water. Housing and all procedures were conducted in accordance with the guidelines of the National Institute of Health and were approved by the University of Michigan Committee on Use and Care of Animals (approval nos. 7835 and 7989). Rats were euthanized by inhalation of carbon dioxide in accordance with the American Veterinary Medical Association guidelines on euthanasia.

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Rainer Benndorf and Michael J. Welsh are retired.

Robert R. Gilmont is deceased. The remaining authors wish to dedicate this study to him.

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Benndorf, R., Gilmont, R.R., Hirano, S. et al. Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues. Cell Stress and Chaperones 23, 813–826 (2018). https://doi.org/10.1007/s12192-018-0890-5

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