PMID- 20386969
OWN - NLM
STAT- MEDLINE
DCOM- 20100806
LR  - 20211020
IS  - 1875-8355 (Electronic)
IS  - 1572-3887 (Linking)
VI  - 29
IP  - 3
DP  - 2010 Apr
TI  - Purification and N-glycosylation analysis of melanoma antigen dopachrome 
      tautomerase.
PG  - 204-12
LID - 10.1007/s10930-010-9241-9 [doi]
AB  - 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.
FAU - Vavricka, Christopher J
AU  - Vavricka CJ
AD  - Department of Biochemistry, Virginia Tech, 111 Engel Hall, West Campus Drive, 
      Blacksburg, VA 24061, USA. cjv@vt.edu
FAU - Ray, Keith W
AU  - Ray KW
FAU - Christensen, Bruce M
AU  - Christensen BM
FAU - Li, Jianyong
AU  - Li J
LA  - eng
GR  - R01 AI019769/AI/NIAID NIH HHS/United States
GR  - AI19769/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - Protein J
JT  - The protein journal
JID - 101212092
RN  - 0 (Antigens, Neoplasm)
RN  - 0 (Peptide Fragments)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.4.21.4 (Trypsin)
RN  - EC 3.5.1.52 (Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase)
RN  - EC 5.3.- (Intramolecular Oxidoreductases)
RN  - EC 5.3.3.12 (dopachrome isomerase)
SB  - IM
MH  - Animals
MH  - Antigens, Neoplasm/*chemistry/genetics/isolation & purification/metabolism
MH  - Glycosylation
MH  - Intramolecular Oxidoreductases/*chemistry/genetics/isolation & 
      purification/metabolism
MH  - Melanoma/*enzymology
MH  - Mice
MH  - Peptide Fragments/chemistry/metabolism
MH  - Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism
MH  - Recombinant Proteins/*chemistry/genetics/isolation & purification/metabolism
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
MH  - Spodoptera/metabolism
MH  - Trypsin/metabolism
EDAT- 2010/04/14 06:00
MHDA- 2010/08/07 06:00
CRDT- 2010/04/14 06:00
PHST- 2010/04/14 06:00 [entrez]
PHST- 2010/04/14 06:00 [pubmed]
PHST- 2010/08/07 06:00 [medline]
AID - 10.1007/s10930-010-9241-9 [doi]
PST - ppublish
SO  - Protein J. 2010 Apr;29(3):204-12. doi: 10.1007/s10930-010-9241-9.