Abstract
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.
Highlights
Tissue factor pathway inhibitor (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 GalNActransferase. rTFPI produced by 293 cells, but not that expressed in Chinese hamster ovary cells or human hepatoma HepG2 cells
The slurry was poured into a 1 X 20-cm column and washedas described [22] except rTFPI/293 labeled with either :"S-Cys/Met or [:'"S]SO,was eluted from the gel and digested withpeptide endoglycosidaseF, and the productws ere applied to a Cln-SepPakin H20.No ["'SS] was eluted from the SepPakwhen rTFPI/293 was labeled with [:'"S]Cys/Met, whereas the "'S was quantitatively elut.ed thatdeoxycholatewasomittedfrom all buffers.Bound T F P I was elutedwith50 mM diethylamine(pH11.5),andtheeluatewas immediatelyneutralized by addition of NaH2P0
Enzymes activities in total extracts of cul- glycoprotein hormonesP, OMCcontains a tripeptideseturedhumanaorticandhuman umbilicalvein endothelial quence, Pro-Val-Lys, 6 residues N-terminal toa glycosylated cells were compared with activities in total extracts of 293 Asn found within rodent adrenocorticotropin (ACTH)
Summary
Unbound oligosaccharides were elutedby washing with lateSV40polyadenylationsequence of a pCMVvector[19]and. Bound oligosaccharides containing terminal cotransfected with pSV2neo into293 cells by electroporation. Trans- 01,4-linked GalNAc were selectively eluted with ml of 20 mM Tris fected cells were selecteads G418-resistant colonies.The same cDNA 5 ) ,150 mM NaCI, 0.02% azide containing 50 mh GalNAc. T F P I expressed in 293 cells (rTFPI/293) and in CHOcells (rTFPI/ GlcN-labeled oligosaccharidesas previously described [25] except the CHO) was purified as described previously [20]. Products were separatedby gel filtration on Bio-Gel P-2. 75-cm' plates were each labeled for 96 h with 1 mCi of NaJ''S]S04 (Amersham Corp.) in 10 ml of sulfate-deficient Dulbecco's modified
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