The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases.
The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.
Lee
Ki-Young
KY
Animal Cell and Medical Glycobiology Laboratory and Liver Cell Signal Transduction Laboratory, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusung, Taejon 305-333, South Korea.
Kim
Hyung Gu
HG
Hwang
Mi Ran
MR
Chae
Jung Il
JI
Yang
Jai Myung
JM
Lee
Young Choon
YC
Choo
Young Kug
YK
Lee
Young Ik
YI
Lee
Sang-Soo
SS
Do
Su-Il
SI
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000037:antagonists & inhibitors*; D014415:Tunicamycin / Q000494:pharmacology; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D014415:Tunicamycin; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase