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Purification and N-Glycosylation Analysis of Melanoma Antigen Dopachrome Tautomerase

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Abstract

Dopachrome tautomerase (DCT) plays a critical role in lowering the oxidative stress resulting from melanogenesis. Levels of DCT are elevated in melanoma cell lines that are especially resistant to chemotherapy and radiation. DCT is processed as a melanoma antigen and is a potential target for immunotherapy. In order to establish a more complete understanding of the role that DCT may play in the etiology and treatment of melanoma skin cancer, isolation of highly pure and properly processed protein is necessary. Purification of native DCT has been problematic due to a hydrophobic transmembrane anchor and interactions with melanin. In this study, DCT was expressed, without its carboxy-terminal transmembrane region using an Sf9 insect cell protein expression system and its recombinant protein was purified by various chromatographic techniques. Analysis of DCT tryptic peptides by MALDI-TOF/TOF determined N-glycosylation as a primary post-translational modification. Our success in the expression of soluble mammalian DCT and the characterization of N-glycosylation sites is a useful reference toward the comprehensive understanding of the structure/function relationship of mammalian DCT.

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Abbreviations

DCT:

Dopachrome tautomerase

MALDI-TOF/TOF:

Matrix-assisted laser desorption/ionization mass spectrometry with tandem time of flight detection

TRP-2:

Tyrosinase-related protein 2 (an alternative name for DCT)

ER:

Endoplasmic reticulum

PMSF:

Phenylmethylsulphonyl fluoride

SDS–PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

DTT:

Dithiothreitol

TFA:

Trifluoroacetic acid

PNGase F:

Peptide N-glycosidase F

Ni–NTA:

Nickel-nitrilotriacetic acid

CID:

Collision induced dissociation

MS:

Mass spectrometry

Da:

Dalton

CAM:

Carboxyamidomethyl

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Acknowledgments

We would like to express our thanks to Dr. Vincent Hearing for generously providing us with DCT antibodies and Dr. Dorothy Bennett for providing us with melan-a -b and -c cell lines that were used in our preliminary studies. We would also like to thank Peipei Liu and Chuansheng Liu at The Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology for their encouragement and comments on the manuscript. Furthermore, we would like to thank Dr. Rich Helm and the Virginia Tech Mass Spectrometry Incubator (http://www.mass.biochem.vt.edu/) for their assistance with the MALDI-TOF/TOF analysis. This work is supported by College of Agricultural Life Sciences, Virginia Tech and NIH grant AI 19769.

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Authors and Affiliations

  1. Department of Biochemistry, Virginia Tech, 111 Engel Hall, West Campus Drive, Blacksburg, VA, 24061, USA

    Christopher J. Vavricka, Keith W. Ray & Jianyong Li

  2. Department of Pathobiology, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Bruce M. Christensen

Authors
  1. Christopher J. Vavricka
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  2. Keith W. Ray
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  4. Jianyong Li
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Correspondence to Christopher J. Vavricka.

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10930_2010_9241_MOESM1_ESM.docx

MALDI-TOF/TOF spectrum of the DCT peptide NECDVCTDELLGAAR, A) with potential fucosylation (additional 146 Da), and B) without modification

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Vavricka, C.J., Ray, K.W., Christensen, B.M. et al. Purification and N-Glycosylation Analysis of Melanoma Antigen Dopachrome Tautomerase. Protein J 29, 204–212 (2010). https://doi.org/10.1007/s10930-010-9241-9

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  • DOI: https://doi.org/10.1007/s10930-010-9241-9

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