Deletion of the cytoplasmic domain of human alpha3/4 fucosyltransferase III causes the shift of the enzyme to early Golgi compartments

The transmembrane domain (TM) and flanking regions of glycosyltransferases (GTs) have been implicated in the localization of these proteins in the Golgi apparatus (GA). alpha3/4 Fucosyltransferase III (FT3wt) (EC 2.4.1.65) is localized in the trans-Golgi and trans-Golgi network (TGN) of baby hamster kidney (BHK) cells and synthesizes Lewis determinants associated with cell adhesion events. We have evaluated the effect of removing the cytosolic domain on the localization of the enzyme and its capacity for synthesizing the Lewis A (Le A) determinant. The mutant where the cytoplasmic domain (Asp-2 to Trp-13) of FT3wt has been deleted (FT3dc) was localized in the Golgi but it was shifted to earlier compartments than FT3wt. The mutant was not detected on the plasma membrane (PM) and glycosylation analysis indicated that FT3dc was transported beyond the endoplasmic reticulum (ER) since complex type glycosylation was observed. Cells expressing FT3dc showed a significantly lower efficiency to synthesize Le A when compared with cells expressing FT3wt, in vivo. This reduction was not due to lower specific activity because both enzyme forms had a similar specific activity in vitro. Therefore, removal of FT3 cytosolic tail caused a shift in enzyme distribution to earlier Golgi compartments concomitant to the decrease of its biosynthetic capacity.     

猪瘟病毒石门强毒株和兔化弱毒疫苗株E2蛋白糖基化位点差异分析

猪瘟病毒强毒株和兔化弱毒疫苗株E2糖蛋白分别含有5个和6个潜在的糖基化位点,其中986N是兔化弱毒疫苗株所特有的。为了分析二者糖基化位点差异及其影响,将去掉信号肽和跨膜区的猪瘟病毒石门强毒株(Shi-men)和兔化弱毒疫苗株(HCLV)E2基因置于蜂素信号肽序列下游,使其在Sf9细胞内表达重组Shimen-E2和HCLV-E2蛋白。结果显示,重组E2蛋白以二聚体的形式分泌表达于细胞培养液中,但二者分子量存在差异。用endo H和PNGase F对纯化后的重组E2蛋白进行去糖基化处理后,二者分子量大小变成一致,证实石门强毒株和兔化弱毒株E2蛋白分子量大小的差异可能是由于糖基化程度的差异所致。对986N糖基化位点进行定点突变后发现,突变后的Shimen-E2与野生型HCLV-E2分子量大小一致,而突变后的HCLV-E2与野生型Shimen-E2分子量大小一致,表明Shimen-E2和HCLV-E2分子量大小的差异的确是由于986N糖基化位点的差异引起的。     

Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites

Heparan N-sulfatase cDNA contains five potential N-glycosylation sites at Asn positions 41, 142, 151, 264, and 413. We used site-directed mutagenesis, substituting the codon of asparagine for glutamine, to eliminate selected glycosylation sites and then performed expression studies in COS-7 cells to determine the influence on the catalytic activity, lysosomal targeting, and glycosylation-phosphorylation of the enzyme. Elimination of site 5 did not affect significantly enzyme activity; elimination of sites 2 and 4 gave a partial reduction, while elimination of sites 1 and 3 resulted in drastic reduction of catalytic activity (25 and 14%, respectively, of normal values), indicating that glycosylation of asparagine 41 and asparagine 151 is essential for catalysis and/or enzyme stability. Wild type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for acquisition of enzyme activity and/or for enzyme stability. Metabolic labeling of each mutant cDNA, transiently transfected into COS cells, showed that enzyme from mutants N142Q, N264Q, and N413Q appeared to be properly folded, as judged by its ability to be proteolytically processed to a lower molecular weight form, while enzyme from mutants N41Q and N151Q did not reach lysosomes. These studies confirm that the five glycosylation sites of heparan N-sulfatase are all functional and show that Asn 41 and Asn 151 have a role in protein folding and/or stability.     

Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells

Mutations in the gene coding for a human ABC transporter protein, ABCC6 (MRP6), are responsible for the development of pseudoxanthoma elasticum. Here, we demonstrate that human ABCC6, when expressed by retroviral transduction in polarized mammalian (MDCKII) cells, is exclusively localized to the basolateral membrane. The human ABCC6 in MDCKII cells was found to be glycosylated, in contrast to the underglycosylated form of the protein, as expressed in Sf9 cells. In order to localize the major glycosylation site(s) in ABCC6, we applied limited proteolysis on the fully glycosylated and underglycosylated forms, followed by immunodetection with region-specific antibodies for ABCC6. Our results indicate that Asn15, which is located in the extracellular N-terminal region of human ABCC6, is the only N-glycosylation site in this protein. The polarized mammalian expression system characterized here provides a useful tool for further examination of routing, glycosylation, and function of the normal and pathological variants of human ABCC6.     

Plasma membrane delivery of the gastric H,K-ATPase: the role of beta-subunit glycosylation

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase beta-subunit (YFP-beta) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase alpha-subunit (CFP-alpha) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-alpha, wild-type YFP-beta, and YFP-beta mutants lacking one or two of the seven beta-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-beta resulted in the plasma membrane localization of the protein, whereas the expressed CFP-alpha was retained intracellularly. When coexpressed, both CFP-alpha and YFP-beta were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-beta prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the beta-subunit are essential for the plasma membrane delivery of the beta-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the beta-subunits from different species, is not critical for plasma delivery of the protein.     

Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module.

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.     

Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster

The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.     

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.     

Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.     

Analyses of catalytic activity and inhibitor binding of human acid beta-glucosidase by site-directed mutagenesis. Identification of residues critical to catalysis and evidence for causality of two Ashkenazi Jewish Gaucher disease type 1 mutations

Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid beta-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles. Comparative studies were conducted with enzymes expressed from natural (spleen and fibroblasts) alleles or from mutagenized cDNAs in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. Mutant cDNAs containing Thr43 to Lys43 (beta-GlcThr43----Lys) and Asp358 to Glu358 (beta-GlcAsp358----Glu) substitutions and two cDNAs containing Ashkenazi Jewish Gaucher disease Type 1 mutations, Arg120 to Gln120 (beta-GlcArg120----Gln) and Asn370 to Ser370 (beta-GlcAsn370----Ser) were expressed and the gene products characterized by enzymatic, immunologic, and inhibitor studies. Genotypes at the acid beta-glucosidase locus in selected Gaucher disease Type 1 patients were determined by allele-specific oligonucleotide hybridization of amplified genomic DNA. Compared with normal, recombinant or natural enzymes expressed from beta-GlcAsn370----Ser alleles had about 2-5-fold decreased specific activity based on CRIM (cross-reacting immunologic material). The beta-GlcArg120----Gln cDNA expressed catalytically inactive CRIM in Sf9; consistent with the 9-fold decreased CRIM-specific activity of the natural enzyme from a beta-GlcArg120----Gln/beta-GlcAsn370----Ser genetic compound. The beta-GlcAsp358----Glu cDNA expressed catalytically inactive CRIM in Sf9 cells. The presence of natural or recombinant enzyme expressed from beta-GlcAsn370----Ser alleles was sufficient to confer 3-5-fold increased IC50 values for deoxynojirimycin, glucosylsphingosine, and N-alkyl-glucosylamine derivatives. Progress curves for inhibition by the slow-tight binding N-alkyl-glucosylamines indicated that the beta-Glc-Asn370----Ser mutation did not alter a conformational change induced by these reaction intermediate analogues. These results provide evidence that the beta-GlcArg120----Gln and beta-GlcAsn370----Ser mutations found in Gaucher disease Type 1 patient genomes are the molecular bases of the enzymatic dysfunction. In addition, the region including Arg120 and that encompassing Asp358 and Asn370 contain residues critical to active site formation or participation in the catalytic mechanism.     

Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312

It has been shown previously that chicken ovalbumin synthesized and secreted in a heterologous cell system is glycosylated at the correct site and that the oligosaccharides at that site, similar to the protein made in hen oviduct, are predominantly of the hybrid type (Sheares, B. T., and Robbins, P. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1993-1997). This site-specific glycosylation of Asn293, but not Asn312, suggested a prominent role for the nascent protein chain rather than the specific cell type in directing the proper attachment of oligosaccharide chains. In the present study, the effect of glycosylation at Asn293 on the glycosylation of Asn312 has been investigated. Using a 20-base oligodeoxynucleotide primer containing a 2-base mismatch, the codon for Asn293 in the chicken ovalbumin gene (AAC) was changed to that for Gln (CAA), thereby preventing glycosylation at amino acid 293. Constructions containing this mutation were transfected into mouse L (tk-) cells which were subsequently labeled with [35S]methionine. Ovalbumin secreted by these cells was recovered by immunoaffinity chromatography and analyzed for the presence of an oligosaccharide attached at Asn312. Treatment of the material with peptide:N-glycosidase F demonstrated that ovalbumin molecules containing Gln substituted for Asn293 were not glycosylated. This further supports our earlier hypothesis that the nascent protein chain is responsible for directing site-specific glycosylation of ovalbumin, and that the presence of an oligosaccharide chain at the first site has no influence on glycosylation at the second site.     

Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5(')-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.     

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.     

N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase

Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.     

The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans

Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry

We have previously determined the carbohydrate structure of human recombinant erythropoietin [Sasaki, H., Bothner, B., Dell, A., & Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076]. The carbohydrate chains are distributed in three N-glycosylation sites and one O-glycosylation site. In order to examine the extent to which protein structure influences glycosylation, we have analyzed the saccharide structures at each glycosylation site (Asn24, Asn38, Asn83, and Ser126) of human recombinant erythropoietin. By high-performance liquid chromatography, we have succeeded in separation of glycopeptides containing different O-linked saccharides to the same peptide backbone. Fast atom bombardment mass spectrometry of the isolated glycopeptides combined with Edman degradation allowed us to elucidate the composition of glycopeptides and the amino acid attachment site. The analysis of glycopeptides and saccharides by fast atom bombardment mass spectrometry and high-performance liquid chromatography provided the following conclusions on N-glycans: (1) saccharides at Asn24 are heterogeneous and consist of biantennary, triantennary, and tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (2) saccharides at Asn38 mainly consist of well-processed saccharides such as tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (3) saccharides at Asn83, on the other hand, are homogeneous in the backbone structure and are composed mainly of tetraantennary without N-acetyllactosaminyl repeats. It was also noted that saccharides at Asn24 are much less sialylated than those at Asn38, although these two glycosylation sites are close to each other. These results clearly indicate that the protein structure and, possibly, the carbohydrate chain at the neighboring site greatly influence glycosylation of a given glycosylation site.     

Glycosylation of the N-terminal potential N- glycosylation sites in the human α1,3- fucosyltransferase V and -VI (hFucTV and -VI)

Human 1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and hFucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced by glutamine. Western blotting analyses demonstrated that both sites in hFucTV were glycosylated, whereas in hFucTVI only one of the sites (Asn91) was glycosylated. All the single mutants and the hFucTVI N46Q/N91Q double mutant exhibited enzyme activities that did not differ considerably from the wt activities. However, the enzyme activity of the hFucTV N60Q/N105Q double mutant was reduced to approximately 40% of the wt activity. In addition, castanospermine treatment diminished the enzyme activity and hence trimming of the N-linked glycans are required for expression of full enzyme activity of both hFucTV and -VI. The present study demonstrates that both of the N-terminal potential N-glycosylation sites in hFucTV and one of the sites in hFucTVI are glycosylated. Individually, their glycosylation does not contribute considerably to expression of enzyme activity. However, elimination of both sites in hFucTV reduces the enzyme activity.