TY - JOUR
T1 - In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2
AU - Lu, Linchao
AU - Varshney, Shweta
AU - Yuan, Youxi
AU - Wei, Hua Xing
AU - Tanwar, Ankit
AU - Sundaram, Subha
AU - Nauman, Mohd
AU - Haltiwanger, Robert S.
AU - Stanley, Pamela
N1 - Publisher Copyright:
© 2023 The Authors
PY - 2023/12
Y1 - 2023/12
N2 - Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[−/−] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[−/−]Slc35c2[−/−] mutants were indistinguishable in skeletal phenotype from Slc35c1[−/−] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[−/−]Slc35c2[−/−] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.
AB - Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[−/−] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[−/−]Slc35c2[−/−] mutants were indistinguishable in skeletal phenotype from Slc35c1[−/−] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[−/−]Slc35c2[−/−] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.
KW - GDP-fucose transporter
KW - Notch signaling
KW - O-fucose glycans
KW - gene deletion
KW - skeletal development
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U2 - 10.1016/j.jbc.2023.105406
DO - 10.1016/j.jbc.2023.105406
M3 - Article
AN - SCOPUS:85177977406
SN - 0021-9258
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
M1 - 105406
ER -