Suppressors of alpha(1,3)fucosylation identified by expression cloning in the LEC11B gain-of-function CHO mutant.
Factors that regulate alpha(1,3)fucosyltransferase activity are important to identify because FUT genes are up-regulated during inflammation, cancer progression, and tumor metastasis. FUT gene activation increases the expression of cell surface oncofetal antigens such as Lewis X, sialyl-Le X and VIM-2. The LEC11B gain-of-function glycosylation mutant displays these antigens and binds E-selectin because it expresses the Fut6B gene that is shown here to lie immediately downstream of the Fut6A gene. A retroviral strategy for expression cloning factors that suppress alpha(1,3)fucosylation in LEC11B cells was developed, and several cDNAs that reverted the LEC11B glycosylation phenotype were isolated. cDNAs that arose most frequently and independently encoded SLC35C2, a putative GDP-fucose transporter (also termed CGI-15 or Ovcov1); Cd63, a tetraspanin membrane protein; and Hdac5, a histone deacetylase. When transfected into LEC11B cells the SLC35C2 cDNA reduced Le X expression with no concomitant suppression of Fut6B gene transcripts. Transfection of the Cd63 cDNA induced low levels of ricin resistance and also did not suppress Fut6B gene transcripts in LEC11B. However, the Hdac5 cDNA induced ricin resistance, reduced fucosylated antigen expression, and essentially eliminated Fut6B gene transcripts. The Hdac5 cDNA isolated by expression cloning encoded the C-terminal region of hamster Hdac5. Overexpression of this partial Hdac5 cDNA or a full-length Hdac5 cDNA, suppressed Fut6B gene transcripts specifically. Thus the expression cloning strategy identified Hdac5 as a trans-acting repressor of the Chinese hamster ovary Fut6B gene and Cd63 and SLC35C2 as novel factors that suppress alpha(1,3)fucosylation by mechanisms unrelated to effects on Fut gene expression.
Chen
Wei
W
Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.
Tang
Jian
J
Stanley
Pamela
P
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006655:Histone Deacetylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D060149:Tetraspanin 30; D014158:Transcription, Genetic / Q000235:genetics
D015703:Antigens, CD; D018076:DNA, Complementary; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D005643:Fucose; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase; D006655:Histone Deacetylases