Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and-VI (hFucTV and-VI) (original) (raw)
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Glycobiology, 2000
The α1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human α1,3/4fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to ∼40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the K m values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved Cterminal N-glycosylation sites for expression of full enzyme activity.
N-Glycosylations of human 1,3-fucosyltransferase IX are required for full enzyme activity
Glycobiology, 2013
Human α1,3-fucosyltransferase IX catalyzes the transfer of L-fucose from guanosine diphosphate-β-L-fucose to Nacetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential Nglycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed 60% of wt activity and N101Q retained 30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2000
All mammalian K-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated. When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme. Results obtained with singly mutated forms of rFuc-TIV indicate that both sites are occupied in mammalian cells. Lack of glycosylation at sites 117^119, 218^220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme. These results show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218^220 playing the major role. However, N-glycosylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of staining for the unglycosylated mutants as is seen for the wild-type rFuc-TIV in transfected cells.
Glycobiology, 2005
The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I,-II,-III, and-IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.
Biases and complex patterns in the residues flanking protein N-glycosylation sites
Glycobiology, 2003
N-Glycosylation, the most common and most versatile protein modification reaction, occurs at the b-amide of the aspargine of the Asn-Xaa-Ser/Thr sequon. For reasons that are unclear, not all such sequons are glycosylated. To find patterns that affect glycosylation, we examined the amino acid residues from the 20th preceding the sequon to the 20th residue following it, using bioinformatics tools. A clean data set of annotated, experimentally verified, glycosylated and nonglycosylated sequons derived from 617 well-defined nonredundant N-and N-,O-glycoproteins listed in SWISS-PROT (June 2002) was used. NXS and NXT sequons were analyzed separately. Although no overt patterns were found to explain sequon occupancy or nonoccupancy, trends for over-or underrepresentation of certain amino acids at particular positions were statistically significant and different in NXS and NXT sequons. In extension of earlier reports, none of the 80 Asn-Pro-Ser/Thr found were glycosylated, and a markedly low level of glycosylation was seen in sequons with Pro at the position following the Ser/Thr. In addition, a general observation was made that the considerable number of glycosylated sequons in the C-terminal 10 residues of glycoproteins suggests that N-glycosylation in these cases may be posttranslational and not cotranslational, as widely accepted.
Glycosylation Pathways in Glycoprotein Biosynthesis
2002
Six purified glycosyltransferases (a P-galactoside cu2 + 6 sialyltransferase, a fi-galactoside ~y2 + 3 sialyltransferase, an a-N-acetylgalactosaminide a2 + 6 sialyltransferase, a fi-galactoside (~1 -+ 2 fucosyltransferase, a P-N-acetylglucosaminide al --+ 3 fucosyltransferase, and a (fucosyl cul + 2) galactoside (~1 ---f 3 N-acetylgalactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Galfil + 4GlcNAcPl+ 2Man, and antifreeze glycoprotein, which contains oligosaccharides with the structure, Gal/X + 3GalNAccul-+ OThr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the...
In vitro glycosylation of proteins: An enzymatic approach
Journal of Biotechnology, 1996
The glycosylation pathway is the most important post-translational modification of a protein and is moreover a highly specific process. The majority of proteins of pharmaceutical interest are glycoproteins. Therefore, it is necessary to identify the composition, the structure, the function and the biosynthesis of the glycoproteins. The present knowledge is described here. In addition, the performed studies about structure-function relationship of the glycoproteins have shown that the oligosaccharide part of a glycoprotein confers important and specific biological roles. Thus, the modification of the structure of the glycan chains can lead to a modification of the activity of the glycoprotein. This phenomenon is encountered at the time of the production of recombinant glycoprotein in a heterologous system. Indeed, the glycosylation profile of a protein is specific to both the host cell and the culture conditions of this cell. Thus, the advantages and the drawbacks of the different host cells used for the glycosylation engineering are presented. In this way, the identification of the different specific enzymes glycosyltransferases and glycosidases involved in the glycosylation pathway is now necessary to improve the production of recombinant glycoprotein. The structure and the characteristics of these enzymes, and more particularly the oligosaccharyltransferase and the galactosyltransferase, are also described.
Biochemistry, 1999
Coagulation factor V (FV) circulates in two forms, FV1 and FV2, having slightly different molecular masses and phospholipid-binding properties. The aim was to determine whether this heterogeneity is due to the degree of glycosylation of Asn 2181. FVa1 and FVa2 were isolated and digested with endoglycosidase PNGase F. As judged by Western blotting, the FVa2 light chain contained two N-linked carbohydrates, whereas FVa1 contained three. Wild-type FV and three mutants, Asn 2181 Gln, Ser 2183 Thr, and Ser 2183 Ala, were expressed in COS1 cells, activated by thrombin, and analyzed by Western blotting. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn 2181 Gln and Ser 2183 Ala mutants contained only the 71 kDa light chain. In contrast, the Ser 2183 Thr mutant gave a 74 kDa light chain. This demonstrated that the third position in the Asn-X-Ser/Thr consensus affects glycosylation efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser 2183 Thr mutant FVa was functionally indistinguishable from plasma-purified FVa1, whereas Asn 2181 Gln and Ser 2183 Ala mutants behaved like FVa2. Thus, the carbohydrate at Asn 2181 impaired the interaction between FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and the position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of Asn 2181 related to the presence of a Ser rather than a Thr at the third position in the consensus sequence of glycosylation.