Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity.
Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs.
Gomez
Alvin
A
Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Nekvindova
Jana
J
Travica
Sandra
S
Lee
Mi-Young
MY
Johansson
Inger
I
Edler
David
D
Mkrtchian
Souren
S
Ingelman-Sundberg
Magnus
M
D016604:Aflatoxin B1 / Q000378:metabolism / Q000633:toxicity; D001216:Asparagine / Q000378:metabolism; D001711:Biotransformation; D018938:Caco-2 Cells; D020134:Catalytic Domain / Q000502:physiology*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D044927:Electron-Transferring Flavoproteins / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D007424:Intracellular Fluid / Q000201:enzymology* / Q000378:metabolism
D044927:Electron-Transferring Flavoproteins; D007211:Indoles; C057812:indoline; D001216:Asparagine; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; C509336:CYP2W1 protein, human; D000072467:Cytochrome P450 Family 2