Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.     

The synthesis of complex-type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicular stomatitis virus G protein.

The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added. The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing. Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine. Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide.     

Biosynthesis of lipid-linked oligosaccharides. Isolation and structure of a second lipid-linked oligosaccharide in Chinese hamster ovary cells.

Previous work has shown that vesicular stomatitis virus-infected Chinese hamster ovary cells contain a major high molecular weight lipid-linked oligosaccharide which is transferred en bloc to protein during the formation of the asparagine-linked complex-type oligosaccharides of the vesicular stomatitis virus G protein (Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722). We now report the characterization of a second, lower molecular weight lipid-linked oligosaccharide. The oligosaccharide portion of this molecule was isolated and its structure was determined by methylation analysis, digestion with exoglycosidases, acetolysis and Smith periodate degradation to be: (formula: see text). Several lines of evidence are presented which indicate that this lipid-linked oligosaccharide is primarily involved in the assembly of the major lipid-linked oligosaccharide rather than in the direct glycosylation of proteins.     

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide

• 1.1. A maximum rate of dolichyl phosphate [¹⁴C]glucose synthesis from 55-day embryos was achieved at 16nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate.
• 2.2. The highest values of [¹⁴C]glucose incorporation from UDP-[¹⁴C]glucose into dolichyl phosphate [¹⁴C]glucose, dolichyl diphosphate [¹⁴C]Glc-oligosaccharides and proteins were reached at 5 min time point of incubation of liver microsomes both from embryos and sows.
• 3.3. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows.
• 4.4. The enzymatic transfer of Glc₃-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides.
• 5.5. One labelled compound was discovered in the Chcl₃-Ch₃Oh-H₂O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[¹⁴C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc₂Man₉Glc₃.

     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Detection of the lipid-linked precursor oligosaccharide of N-linked protein glycosylation in Drosophila melanogaster

The presence of a glycan of the same molecular size as the lipid linked precursor oligosaccharide (Glc₃Man₉GlcNAc₂) of the N-linked protein glycosylation pathway in mammalian cells has been detected in a glycolipid fraction of cultured Drosophila melanogaster cells. Oligosaccharide sequencing studies were consistent with the existence of a glucosylated high mannose containing structure, which may be the common precursor for N-linked protein glycosylation in insect cells.     

Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates.

Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.

Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.     

Stimulation of lipid-linked oligosaccharide assembly during oviduct differentiation

Regulation of dolichyl phosphate-linked oligosaccharide assembly has been studied during the course of diethylstilbestrol-induced chick oviduct differentiation. Oviduct membranes from treated chicks form 4.6 times as much GlcNAc-P-P-Dol and GlcNAc2-P-P-Dol upon incubation with UDP-[14C]GlcNAc and MgCl2 than do membranes from untreated chicks. Assembly of oligosaccharide-lipid was studied by incubation of membranes with purified exogenous [14C]GlcNAc2-P-P-Dol and GDP-Man. Man transfer required a divalent cation (10 mM Mg2+) and detergent (0.5% Nonidet P-40 is optimal) and occurs in the presence of amphomycin (500 micrograms/ml). The apparent Km for GDP-Man is 1 microM and for [14C]GlcNAc2-P-P-Dol is 0.45 microM. The products are a series of sequentially formed dolichyl pyrophosphate-linked saccharides up to Man5GlcNAc2, the first of which is Man beta 1,4GlcNAc2. The same products are formed either in the presence or absence of amphomycin. Conversion of GlcNAc2-P-P-Dol to higher oligosaccharides is stimulated 3-fold by estrogen treatment of chicks. Similarly, the conversion of partially purified exogenously added Man beta-[14C]GlcNAc2-P-P-Dol is 4.6-fold higher after diethylstilbestrol treatment.     

Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides

Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.     

Inhibition of protein synthesis also inhibits synthesis of lipid-linked oligosaccharides.

Studies were initiated to determine whether the formation of lipid-linked oligosaccharides was coupled to the synthesis of protein. Canine kidney cells were grown with [2-3H]mannose or [3H]leucine in the presence of cycloheximide or puromycin and the effect of these inhibitors on the synthesis of proteins and lipid-linked oligosaccharides was measured. In all cases, the inhibition of protein synthesis resulted in a substantial inhibition in the incorporation of mannose into the lipid-linked oligosaccharides, although the synthesis of mannosyl-phosphoryl-dolichol was only slightly inhibited. Cycloheximide had no effect on the in vitro incorporation of mannose into lipid-linked oligosaccharides when GDP-[14C]mannose was incubated with aorta microsomal preparations. The inhibition of lipid-linked oligosaccharides was apparently not due to a decrease in the amount of glycosyltransferases as a result of protein degradation in the absence of protein synthesis, nor was it the result of a more rapid degradation of lipid-linked oligosaccharides. The inhibition also did not appear to be due to limitations in the available dolichyl-phosphate. The results suggest that the formation of lipid-linked oligosaccharides may be regulated by end product inhibition.     

Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins.

Some asparagine and glutamine residues in proteins undergo deamidation to aspartate and glutamate with rates that depend upon the sequence and higher-order structure of the protein. Functional groups within the protein can catalyze this reaction, acting as general acids, bases, or stabilizers of the transition state. Information from specific proteins that deamidate and analysis of protein sequence and structure data bases suggest that asparagine and glutamine lability has been a selective pressure in the evolution of protein sequence and folding. Asparagine and glutamine deamidation can affect protein structure and function in natural and engineered mutant sequences, and may play a role in the regulation of protein folding, protein breakdown, and aging.     

Abnormal lipid-linked oligosaccharides in class E Thy-1-negative mutant lymphomas.

The glycosylation defect of Thy-1-mutant lymphomas of the class E complementation group has been identified as a block in the synthesis of the lipid-linked oligosaccharide precursor of the asparagine-linked oligosaccharides of glycoproteins. Two major lipid-linked oligosaccharides were isolated from the mutant cells. Both oligosaccharides were smaller than the lipid-linkid oligosaccharides of wild-type lymphomas and, in contrast to the lipid-linked oligosaccharides isolated from wild-type cells, both were resistant to digestion with endoglycosidase H. The oligosaccharides of newly synthesized polypeptides in class E Thy-1-cells were also resistant to endoglycosidase H digestion, providing strong evidence that they are derived from the abnormal lipid-linked oligosaccharides.     

Glycoprotein biosynthesis in plants. Demonstration of lipid-linked oligosaccharides of mannose and N-acetylglucosamine.

Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends...     

The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases

We recently presented a model for site-specific protein N-glycosylation in Trypanosoma brucei whereby the TbSTT3A oligosaccharyltransferase (OST) first selectively transfers biantennary Man(5)GlcNAc(2) from the lipid-linked oligosaccharide (LLO) donor Man(5)GlcNAc(2)-PP-Dol to N-glycosylation sequons in acidic to neutral peptide sequences and TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to any remaining sequons. In this paper, we investigate the specificities of the two OSTs for their preferred LLO donors by glycotyping the variant surface glycoprotein (VSG) synthesized by bloodstream-form T. brucei TbALG12 null mutants. The TbALG12 gene encodes the α1-6-mannosyltransferase that converts Man(7)GlcNAc(2)-PP-Dol to Man(8)GlcNAc(2)-PP-Dol. The VSG synthesized by the TbALG12 null mutant in the presence and the absence of α-mannosidase inhibitors was characterized by electrospray mass spectrometry both intact and as pronase glycopetides. The results show that TbSTT3A is able to transfer Man(7)GlcNAc(2) as well as Man(5)GlcNAc(2) to its preferred acidic glycosylation site at Asn263 and that, in the absence of Man(9)GlcNAc(2)-PP-Dol, TbSTT3B transfers both Man(7)GlcNAc(2) and Man(5)GlcNAc(2) to the remaining site at Asn428, albeit with low efficiency. These data suggest that the preferences of TbSTT3A and TbSTT3B for their LLO donors are based on the c-branch of the Man(9)GlcNAc(2) oligosaccharide, such that the presence of the c-branch prevents recognition and/or transfer by TbSTT3A, whereas the presence of the c-branch enhances recognition and/or transfer by TbSTT3B.