Endogenous Glycosyltransferases Research Articles

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

Direct Visualization of Live Zebrafish Glycans via Single-Step Metabolic Labeling with Fluorophore-Tagged Nucleotide Sugars.

Dynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two-step labeling sequence is required, which suffers from the tissue-penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogues of the nucleotide sugars. Injecting fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables systematic imaging of sialylation and fucosylation in live zebrafish embryos at distinct developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

Promoter Choice Impacts the Efficiency of Plant Glyco-Engineering.

Glyco-modulation of therapeutic proteins produced in plants has shown great success. Plant-based expression platforms for tailored human-like N-glycosylation are based on the overexpression of foreign genes. However, drawbacks such as protein miss targeting, interference with endogenous glycosyltransferases, or with plant development hamper the widespread use of the technology. Here a technique that facilitates the generation of recombinant proteins with targeted N-glycosylation at high homogeneity is described. It is focused on the synthesis of human-type β1,4-galactosylation by the overexpression of the human β1,4-galactosyltransferase (GalT) in Nicotiana benthamiana. A GalT construct that targets the enzyme to the required late Golgi compartment (ST GalT) is transiently co-expressed with two pharmaceutically relevant glycoproteins. The impact of eight promoters driving the expression of ST GalT is evaluated by mass spectrometry (MS) -based analyses. It is shown that five promoters (amongst them high expressors) induce aberrant non-human glycosylation. In contrast, three promoters, considered as moderately active, regulate gene expression to levels leading to an improved efficiency of di-galactosylation (and subsequent sialylation) on the reporter proteins. The results point to the importance of promoter choice for optimizing glycan engineering processes.

Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology.

Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.

A non-enzymatic function of Golgi glycosyltransferases: Mediation of Golgi fragmentation by interaction with non-muscle myosin IIA

The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (β-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases--C2GnT-L and β-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by β-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases.

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

The Regulation of Ganglioside GM3 Synthesis

Glycosphingolipids (GSLs) exist in the outer leaflet of the plasma membrane, where they form lipid microdomains that function as platforms for the regulation of trans-membrane signal transduction. In mammals, complex GSLs differing in the number and/or type of sugar species are produced in a cell-type specific manner, and the variety of glycan structures in GSLs are believed to determine specific cell functions. The glycan moieties of GSLs are synthesized at the luminal side of the Golgi apparatus by multiple Golgi-resident glycosyltransferases. Since the expression levels of most endogenous glycosyltransferases are relatively low, their detection is generally difficult. Nevertheless, we have succeeded in detecting endogenous mouse GM3 synthase (GM3S), the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. Mouse GM3S (mGM3S) has three isoforms (M1-GM3S, M2-GM3S, and M3-GM3S), each with a distinct length in its NH2-terminal cytoplasmic tail. These isoforms are produced by leaky scanning from two mRNA variants, mGM3Sa and mGM3Sb. M1-GM3S is stably localized in the endoplasmic reticulum (ER), as a result of retrograde transport signals (arginine [R]-based motifs); consequently, its in vivo GM3 synthesis activity is very low compared with that of other isoforms. In contrast, both M2-GM3S and M3-GM3S are localized in the Golgi apparatus, yet each exhibits a distinct intracellular fate. M2-GM3S is rapidly degraded in the lysosomes, whereas M3-GM3S is retained in the Golgi apparatus. A system that produces GM3S isoforms having such distinct characteristics is likely to be of critical importance in the regulation of GM3 biosynthesis under various pathological and physiological conditions.

Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes approximately 3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal(2)GlcNAc(2)Man(3)GlcNAc(2)N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

Butyrate induces sLex synthesis by stimulation of selective glycosyltransferase genes

Sialyl Lewisx (sLex) is an important tumor-associated carbohydrate antigen present on the cell surface glycoconjugates involved in leukocyte migration and cancer metastasis. We report the formation of sLex epitope in butyrate-treated human pancreatic adenocarcinoma cells expressing MUC1 and core 2 N-acetylglucosaminyltransferase (C2GnT). Butyrate treatment stimulates not only the transgene but also a group of endogenous glycosyltransferase genes involved in the synthesis of sLex. Current finding raises a concern about the proposed use of butyrate as a cancer therapeutic agent.

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.

The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides

The free-living nematode Caenorhabditis elegans is a relevant model for studies on the role of glycoconjugates during development of multicellular organisms. Several genes coding for glycosyltransferases involved in the synthesis of N- and O-linked glycans have already been isolated, but, apart from repetitive dimers of glycosaminoglycans, no detailed structure of either type of component has been published so far. This study aimed to establish the structures of the major O-glycans synthesized by C. elegans to give an insight into the endogenous glycosyltransferase activities expressed in this organism. By the use of NMR and MS, we have resolved the sequence of seven of these components that present very unusual features. Most of them were characterized by the type-1 core substituted on Gal and/or GalNAc by (β1–4)Glc and (β1–6)Glc residues. Another compound exhibited the GalNAc(β1–4)N-acetylglucosaminitol sequence in the terminal position, to which was attached a tetramer of β-Gal substituted by both Fuc and 2-O-methyl-fucose residues. Our experimental procedure led also to the isolation of glycosaminoglycan-like components and oligomannosyl-type N-glycans. In particular, the data confirmed that C. elegans synthesizes the ubiquitous linker sequence GlcA(β1–3)Gal(β1–3)Gal(β1–4)Xyl.

The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides.

The free-living nematode Caenorhabditis elegans is a relevant model for studies on the role of glycoconjugates during development of multicellular organisms. Several genes coding for glycosyltransferases involved in the synthesis of N- and O-linked glycans have already been isolated, but, apart from repetitive dimers of glycosaminoglycans, no detailed structure of either type of component has been published so far. This study aimed to establish the structures of the major O-glycans synthesized by C. elegans to give an insight into the endogenous glycosyltransferase activities expressed in this organism. By the use of NMR and MS, we have resolved the sequence of seven of these components that present very unusual features. Most of them were characterized by the type-1 core substituted on Gal and/or GalNAc by (beta1-4)Glc and (beta1-6)Glc residues. Another compound exhibited the GalNAc(beta1-4)N-acetylglucosaminitol sequence in the terminal position, to which was attached a tetramer of beta-Gal substituted by both Fuc and 2-O-methyl-fucose residues. Our experimental procedure led also to the isolation of glycosaminoglycan-like components and oligomannosyl-type N-glycans. In particular, the data confirmed that C. elegans synthesizes the ubiquitous linker sequence GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl.

Stable transfection of human β-1,4N-acetylgalactosaminyltransferase and α-2,8-sialyltransferase cDNAs in C6 rat glioma cells induces modifications in ganglioside metabolism

Ganglioside distribution in cells undergoes deep modifications during physiological and pathological events, possibly depending on the activity of glycosyltransferases involved in their biosynthesis. To understand how the ganglioside pattern can be altered by the selective expression of specific glycosyltransferases, C6 rat glioma cell line was stably transfected with two human glycosyltransferase cDNAs: β-1,4N-acetylgalactosaminyltransferase (GaINAcT) and a-2,8-sialyltransferase (ST-II). GaINAcT and ST-II are key enzymes in ganglioside biosynthesis; whereas ST-II synthesizes GD 3 , precursor of the b pathway, GaINAcT produces GM 2 , GD 2 and asialo-GM 2 and it is, therefore, involved in a, b and asialo pathways. C6 cells were subjected to three independent transfections: one with a construct containing GaINAcT cDNA, one with a construct containing the ST-II cDNA, and one with both constructs simultaneously. Whereas control cells present mainly N-acetyl- and N-glycolyl-GM 3 , selected transfected clones show more complex ganglioside profiles: GaINAcT-expressing cells are enriched in the a series gangliosides, ST-II-expressing cells synthesize the b series species, cells expressing contemporarily the two glycosyltransferases produce gangliosides of both series. Furthermore, among the selected clones, expression of GaINAcT and ST-II correlates with changes in the ST-I and ST-IV activities, indicating that the switching on of the biosynthetic enzymes we investigated influences the activity of endogenous glycosyltransferases, possibly through the modification of the amount of their substrates or products.

Kiwifruits (Actinidia deliciosa) transformed with a Vitis stilbene synthase gene produce piceid (resveratrol-glucoside).

A stilbene synthase gene was isolated from three Vitis spp. (V. vinifera, V. labrusca and V. riparia). These genes were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into kiwifruit (Actinidia deliciosa) plants by Agrobacterium-mediated gene transfer. The introduced gene(s) were expressed and piceid (resveratrol-glucoside) rather than resveratrol was produced in the leaves of the transformants. Resveratrol produced by the action of the integrated gene(s) seems to be metabolized into piceid by an endogenous glycosyltransferase. Among the transformants obtained, the highest piceid content in the young leaves was 182 μg/g fresh weight. Although these transformants did not show resistance against Botrytis cinerea, which causes gray-mould disease, the fruits may have some beneficial effects on human health.

Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels.

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

Alteration of oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases.

The alteration of oligosaccharide structures through genetic manipulation of glycosyltransferase activities is now a reality. It is apparent that this technique has greater consequences on oligosaccharide structure when an exogenous enzyme is introduced into cells, and in particular when this enzyme is responsible for a terminal glycosylation step. By contrast, only one study has examined the effects of overexpressing an endogenous glycosyltransferase, in which there was no detectable effect on glycosylation. However, there are still other key regulatory biosynthetic enzymes, such as GlcNAc transferase V and beta 1,3 GlcNAc transferase, whose overexpression may alter glycosylation. Both of these enzymes are required for the biosynthesis of polylactosaminoglycans (polymers of N-acetyllactosamine disaccharides), and their elevation in tumor cells correlates with increased expression of polylactosaminoglycans. Recently, the gene encoding GlcNAc transferase V has been isolated, but its transfection into cells and characterization of the resulting oligosaccharides awaits further study. Alternate strategies for modifying oligosaccharide structures could involve the introduction of more than one glycosyltransferase into cells to ensure the availability of biosynthetic intermediates. Alternatively, the disruption of specific glycosyltransferase genes by homologous recombination could be used to eliminate competing glycosyltransferases that act on a common substrate. Although oligosaccharide biosynthesis is directly dependent upon the presence or absence of specific glycosyltransferases, other factors also contribute to glycosylation. For example, the transport rate of a glycoprotein through the endoplasmic reticulum and Golgi complex, the levels of processing glycosidases, the availability of substrates, the host cell, and ultimately, the peptide backbone of the particular glycoprotein of interest are important contributors to the final outcome of oligosaccharide structure. Despite these complications, further study into the manipulation of glycosyltransferase genes may ultimately allow the controlled and predictable biosynthesis of glycoprotein sugar chains.

Medial-Golgi retention of N-acetylglucosaminyltransferase I. Contribution from all domains of the enzyme.

We have previously shown that the transmembrane domain and flanking residues of beta-1,2-N-acetylglucosaminyltransferase I (GnTI) can localize a hybrid molecule to medial-Golgi cisternae (Burke, J., Pettitt, J. M., Schachter, H., Sarkar, M., and Gleeson, P.A. (1992) J. Biol. Chem. 267, 24433-24440). Here, we have further examined the contribution of the cytoplasmic tail, transmembrane domain, and the catalytically active, luminal domain of GnTI in medial-Golgi localization, by analyzing the localization of hybrid molecules stably expressed in murine cells. In contrast to wild-type GnTI, which was efficiently localized to the medial-Golgi and not detected at the cell surface, hybrid molecules containing any two of the three domains of GnTI were localized to the medial-Golgi and were also present at low levels at the cell surface. Hybrid molecules containing only the transmembrane domain or the luminal domain of GnTI showed partial Golgi retention together with an increased level of cell surface expression compared with molecules containing two GnTI domains. The cytoplasmic tail independently was unable to retain reporter sequences to the Golgi but increased the ability of constructs containing either the luminal or transmembrane domain of GnTI to localize to the Golgi apparatus. Therefore, all three domains of GnTI contribute significantly to medial-Golgi localization. Furthermore, GnTI hybrid molecules showing increased cell surface expression were more readily extracted in a low salt buffer, suggesting that Golgi localized GnTI differs in physicochemical properties from cell surface GnTI. Based on an aggregation model of localization, we propose that Golgi retention of these hybrid molecules is mediated by the interaction of their GnTI domains with the corresponding domains of endogenous glycosyltransferase aggregates within the Golgi membranes of the transfected cell.