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Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines

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Abstract

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from RAGE−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope.

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Acknowledgements

We are grateful to Meaghan E. Killeen and JD Lynam for their technical support, to Dr. Takuo Watanabe and Dr. Hiroshi Yamamoto of Kanazawa University Graduate School of Medical Science (Kanazawa, Japan) for esRAGE-specific monoclonal antibody and human RAGE expression vectors (Yonekura et al. 2003), to Dr. Ronald Koslowski of Dresden University of Technology (Dresden, Germany) for providing the R3/1 rat type 1 pneumocyte-derived cell line (Koslowski et al. 2004), to Dr. Edwin Ades (National Center for Infectious Diseases, Atlanta, Georgia, USA) for the HMEC-1 cells (Ades et al. 1992), to Dr. Tim Oury of the University of Pittsburgh and Dr. Angelika Bierhaus of the University of Heidelberg (Heidelberg, Germany) for providing tissue from RAGE knockout mice, and to Dr. Billy Day and Dr. Manimalha Balasubramani of the University of Pittsburgh Genomics and Proteomics Core Facility for assistance with RAGE mass spectroscopy.

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Authors and Affiliations

  1. Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

    Julia V. Gefter, Angel L. Shaufl, Mitchell P. Fink & Russell L. Delude

  2. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

    Julia V. Gefter & Russell L. Delude

  3. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

    Mitchell P. Fink

  4. 615 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA

    Russell L. Delude

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  1. Julia V. Gefter
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  2. Angel L. Shaufl
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Correspondence to Russell L. Delude.

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This work was supported by NIH grants R01 GM37631 and GM68481.

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Gefter, J.V., Shaufl, A.L., Fink, M.P. et al. Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines. Cell Tissue Res 337, 79–89 (2009). https://doi.org/10.1007/s00441-009-0791-0

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