Abstract
Each of the five human alpha1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human beta-trace protein (beta-TP) as a secretory reporter glycoprotein. In order to study their in vivo properties for the transfer of peripheral Fuc onto N-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All fucosyltransferases were found to peripherally fucosylate 19-52% of the diantennary beta-TP N-glycans, and all enzymes were capable of synthesizing the sialyl LewisX (sLex) motif. However, each enzyme produced its own characteristic ratio of sLex/Lex antennae as follows: FT7 (only sLex), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4 (1:7). Fucose transfer onto beta-TP N-glycans was low in FT3 cells (11% of total antennae), whereas the values for FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%, respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Lex on asialo diantennary N-glycans and mono-Lex with monosialo chains. In contrast, FT6 forms mostly alpha1,3-difucosylated chains with no, one, or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium in significant amounts, whereas FT7 and FT5 were found to be largely resistant toward proteolysis. Studies on engineered soluble variants of FT6 indicate that these forms do not significantly contribute to the in vivo fucose transfer activity of the enzyme when expressed at activity levels comparable to those obtained for the wild-type Golgi form of FT6 in the recombinant host cells.
Highlights
Each of the five human ␣1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human -trace protein (-TP) as a secretory reporter glycoprotein
In order to study their in vivo properties for the transfer of peripheral Fuc onto N-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein
In Vitro and in Vivo Synthesis of Lex and sialyl LewisX (sLex) Motifs in -TP N-Glycans by Recombinant Human FT6 —Human -TP expressed from BHK-21 cells in the absence of recombinant FTs contains almost exclusively diantennary complex-type II oligosaccharides at its two N-glycosylation sites
Summary
Materials—Peptide-N4-(N-acetyl--D-glucosaminyl)asparagine amidase F (from Flavobacterium meningosepticum) and endo--N-acetyl-Dglucosaminidase H (from Streptomyces plicatus), both recombinant from Escherichia coli, were bought from Boehringer Mannheim, Vibrio cholerae sialidase was purchased from Calbiochem (La Jolla, CA), DNazol and Tri-Reagent were from Molecular Research Center, Inc. (Cincinnati, OH), Jetsorb was obtained from Genomed (Bad Oeynhausen, Germany), fetal bovine serum was purchased from ITM (Munich, Germany), GDP-[14C]Fuc (285 Ci/mol) was from Amersham Pharmacia Biotech (Braunschweig, Germany), and bovine fetuin, GDP-Fuc, and G418 sulfate were bought from Sigma. A second fragment was obtained by PCR of genomic DNA from human A172 cells, which was prepared as described [36], using the sense mutagenesis primer 5Ј-GGG TCC CGC TTC GTC GAC AGC ACA GGG ACC (SalI site underlined) and the antisense primer 5Ј-GAA GCT TCA GAT CTA CGA GTC CTT AGG at identical PCR conditions except that a 2-min elongation time was used. Both fragments were cloned as described above into pCR3 that contains a NotI site at the 3Ј-end of the multiple cloning site, yielding the vectors pCRIL43.1 and pCRFucN2C. No 3-substituted GlcNAc was detected by this procedure excluding the presence of type I motifs/ Lea structures
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