Core glycosylation of cytochrome P-450(arom). Evidence for localization of N terminus of microsomal cytochrome P-450 in the lumen.
It was found that cytochrome P-450(arom) purified from human placenta microsomes is glycosylated, and the sugar chain was cleaved with endoglycosidase H (Endo H). The core glycosylation of P-450(arom) was examined with two heterologous expression systems, cultured insect cells and in vitro translation system. The P-450(arom) protein expressed in the insect cells was glycosylated, and the sugar chain was sensitive to Endo H. It was also glycosylated when translated with the wheat germ cell-free system in the presence of rough microsomal membrane, and the sugar chain could be removed by Endo H treatment. Since the P-450(arom) molecule has two potential glycosylation sites (Asn-12 and Asn-180), we replaced each of the 2 asparagine residues with alanine by site-directed mutagenesis and examined the glycosylation of the two mutant proteins in the cell-free system. The core glycosylation did not occur when the Asn-12 residue was mutated, whereas the mutant protein with modified Asn-180 residue was glycosylated. These results demonstrated that the potential glycosylation site (Asn-12) in the N-terminal portion of P-450(arom) is the site of glycosylation. We conclude that the N terminus of P-450(arom) is translocated across the endoplasmic reticulum membrane to be glycosylated at the luminal side.
Shimozawa
O
O
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Sakaguchi
M
M
Ogawa
H
H
Harada
N
N
Mihara
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D006031:Glycosylation; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001141:Aromatase