TY - JOUR
T1 - Suppressors of α(1,3)fucosylation identified by expression cloning in the LEC11B gain-of-function CHO mutant
AU - Chen, Wei
AU - Tang, Jian
AU - Stanley, Pamela
N1 - Funding Information:
Accession number: CHO Hdac5 AY145846. This work was supported by a grant from the National Institutes of Health (RO1 CA30645 to P.S.) and by partial support from Albert Einstein Cancer Center Grant PO1 13330. The authors thank Dr. Aimin Zhang for helpful discussions and reagents and Subha Sundaram for excellent technical assistance.
PY - 2005/3
Y1 - 2005/3
N2 - Factors that regulate α(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 α(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 α(1,3)fucosylation by mechanisms unrelated to effects on Fut gene expression.
AB - Factors that regulate α(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 α(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 α(1,3)fucosylation by mechanisms unrelated to effects on Fut gene expression.
KW - Dominant CHO mutant
KW - Expression cloning
KW - Le X suppression
KW - α(1,3)fucosyltransferase
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U2 - 10.1093/glycob/cwi011
DO - 10.1093/glycob/cwi011
M3 - Article
C2 - 15525819
AN - SCOPUS:14844334185
SN - 0959-6658
VL - 15
SP - 259
EP - 269
JO - Glycobiology
JF - Glycobiology
IS - 3
ER -