The asparagine-linked oligosaccharides on tissue factor pathway inhibitor terminate with SO4-4GalNAc beta 1, 4GlcNAc beta 1,2 Mana alpha.
Tissue factor pathway inhibitor (TFPI) produced by endothelial cells contains sulfated Asn-linked oligosaccharides. We have determined that greater than 70% of the oligosaccharides on recombinant TFPI expressed in 293 cells terminate with the sequence SO4-4GalNAc beta 1, 4GlcNAc beta 1, 2Man alpha. Oligosaccharides terminating with this sequence have previously been described on lutropin, thyrotropin, and pro-opiomelanocortin: glycoproteins synthesized in the anterior pituitary. A GalNAc-transferase that recognizes the tripeptide motif Pro-Xaa-Arg/Lys 6-9 residues N-terminal to Asn glycosylation sites accounts for the specific addition of GalNAc to the oligosaccharide acceptor on these glycoproteins, whereas a GalNAc beta 1,4GlcNAc beta 1, 2Man alpha-4-sulfotransferase accounts for the addition of sulfate. The sulfated oligosaccharides present on these hormones are responsible for their rapid clearance from plasma by a receptor in hepatic reticuloendothelial cells. GalNAc- and sulfotransferase activities with the same properties as those expressed in the pituitary are detected at high levels in 293 cells and at lower levels in endothelial cells. Chinese hamster ovary (CHO) cells do not contain detectable levels of either transferase and rTFPI expressed in CHO cells does not contain sulfated Asn-linked oligosaccharides. TFPI contains the sequence Pro-Phe-Lys, 9 residues N-terminal to the glycosylation site at position 228; this tripeptide may act as the recognition sequence for the GalNAc-transferase. rTFPI produced by 293 cells, but not that produced by CHO cells, is bound by the receptor on hepatic reticuloendothelial cells suggesting the sulfated structures play a role in the biologic behavior of TFPI.
Smith
P L
PL
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri.
Skelton
T P
TP
Fiete
D
D
Dharmesh
S M
SM
Beranek
M C
MC
MacPhail
L
L
Broze
G J
GJ
Baenziger
J U
JU
D000085:Acetates / Q000737:chemistry; D019342:Acetic Acid; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000378:metabolism; D008657:Metabolic Clearance Rate; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000085:Acetates; D008074:Lipoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D001216:Asparagine; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C072136:glycoprotein hormone-specific N-acetylgalactosamine transferase; C498300:CHST9 protein, human; C092527:N-acetylgalactosamine-4-sulfotransferase; D015238:Sulfotransferases; D001619:beta-N-Acetylhexosaminidases; D019342:Acetic Acid