Originally published In Press as doi:10.1074/jbc.R400036200 on December 7, 2004
J. Biol. Chem., Vol. 280, Issue 5, 3121-3124, February 4, 2005
Minireview
Unraveling the Mechanism of Protein N-Glycosylation*
Aixin Yan and
William J. Lennarz
From the
Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794
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INTRODUCTION
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Asparagine-linked glycosylation is the most ubiquitous protein co-translational modification in the endoplasmic reticulum (ER).1 The enzyme that catalyzes this process is called oligosaccharyl transferase (OT). It catalyzes the transfer of an oligosaccharyl moiety (Glc3Man9GlcNAc2) from the dolichol-linked pyrophosphate donor to the side chain of Asn within a consensus sequence of Asn-X-Thr/Ser, where X can be any amino acid residue except for Pro (1–3). This modification serves as a primary determinant for specific molecular recognition as well as protein folding and stability (4, 5) and therefore is an essential and highly conserved protein modification pathway in eukaryotic cells. It has been established in both yeast and higher eukaryotic organisms that this enzyme exists as a heteromeric, multisubunit complex in the ER membrane.
In the last decade, because of the facility of yeast genetics, genes encoding 9 OT subunits in Saccharomyces cerevisiae have been cloned and identified (for review see Ref. 2 and earlier studies cited therein). Among them, the five genes encoding Ost1p, Ost2p, Stt3p, Wbp1p, and Swplp are essential for the viability of the cell; the OST4 gene is essential for growth of the cell at 37 °C but not at 25 °C; Ost3p, Ost5p, and Ost6p subunits are not essential for the viability of the yeast cell but are required for maximal enzyme activity.
At present, many mammalian OT subunit proteins have also been identified, and some of them share high sequence identity and similarity with yeast homologs. Four of them were isolated from highly purified and active enzyme fractions: ribophorin I (homolog of yeast Ost1p), ribophorin II (homolog of yeast Swp1p), OST48 (homolog of yeast Wbp1p), and DAD1 (homolog of yeast Ost2p) (6–10). Recently STT3-A and STT3-B (homologs of yeast Stt3p), N33 and implantation-associated protein (homologs of yeast Ost3p and Ost6p, respectively), as well as OST4 (homolog of yeast Ost4p) have been characterized by genome-wide searches (11). These proteins have been demonstrated to be assembled together with ribophorin I, ribophorin II, OST48, and DAD1 into a multimeric complex similar to the yeast OT (11).
Although genetic studies have yielded considerable information on this enzyme complex, one of the fundamental questions, the enzymatic mechanism of N-glycosylation, has remained unanswered. Specifically, the substrate recognition and/or catalytic sites, the role of each of the subunits, and how they interact structurally has continued to be obscure. In this review, we focus on explorations developed within the past 5 years to clarify the mechanism of this highly conserved protein modification pathway as well as the function of each of the subunits.
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The Nine-yeast OT Subunits Form a Catalytically Active Complex by Specific Interactions with Each Other
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The concept that OT functions as an enzyme complex originated from the earlier biochemical studies where it was demonstrated that in a variety of organisms multiple components were detected in the purified fraction that exhibited highest specific OT activity (6, 8, 9, 12–14). Thus far, it is not certain how these subunits interact to form a catalytically active complex in vivo. Genetic screens to characterize a particular gene with respect to its capability of suppressing the phenotype caused by a null or by a mutation in another gene have provided a few clues to the interrelationship of some of the OT subunits (15–17). Combining these genetic findings with the results of biochemical co-immunoprecipitation experiments, a concept was developed that OT is composed of three subcomplexes: Ost1p-Ost5p, Ost3p-Stt3p-Ost4p, and Ost2p-Wbp1p-Swp1p (17).
However, recently application of a cross-linker, dithiobissuccinimidylpropionate, which bears a spacer arm of 12 Å, to study the interrelationship of any two of all of the yeast OT subunits has yielded a model that is not completely consistent with the above "subcomplex" model (18). In this study, we found that Ost3p and Ost6p cannot co-immunoprecipitate with each other suggesting that they do not exist in the same complex, but they showed exactly the same interaction pattern with all other OT subunits. This finding combined with a recent split ubiquitin study led to our proposal that the two isoforms of OT complex exist in the ER membrane and differ only in the presence of either Ost3p or Ost6p.2 Fig. 1 demonstrates the structural interrelationship of the eight OT subunits in the complex containing Ost3p. Ost6p is proposed to be located in the corresponding position in another form of the OT complex that lacks Ost3p. In this model, five essential gene products in OT complex were found to be within a distance of 12 Å of each other. Two low molecular weight subunits, Ost4p and Ost5p, were shown to interact with only a restricted number of subunits and were proposed to locate closely to Stt3p and Ost1p, respectively. Most interestingly, Ost1p was found to be cross-linked to all of the other eight components (18) and therefore was placed in the core of the OT complex, probably playing an important role in the assembly of the enzyme complex (Fig. 1).
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FIG. 1.
A model of the interrelationship of yeast OT subunits detected by cross-linking studies. Five essential gene products (shown as green) are located within 12 Å of each other. Ost4p and Ost5p (light blue) are found to interact only with a restricted number of subunits. Ost3p and Ost6p (blue) are present in the complex alternatively. The length of the arrows does not indicate the distance between the two proteins.
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Mechanistic Complexities of OT: Structural Integrity and Correct Anchoring of Enzyme Complex in ER Membrane Is Prerequisite for Functional Activity
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It is clear that each of the nine gene products of yeast OT contribute to the efficiency of the enzymatic glycosylation process because null or mutations on each of these genes caused reduction or loss of OT activity in vivo and in vitro (2). Why does the OT enzyme require so many different subunits? Co-translational glycosylation of polypeptides is a very complex process. Among the mechanistic complexities are the requirements to ensure the active site recognizes glycosylatable consensus sequences in the rapidly growing polypeptide chain, to activate the Asn amide nitrogen for nucleophilic attack, and to ensure the rapid product discharge from the active site. In addition to these catalytic events of N-glycosylation per se, it is likely that the active site of OT must be directed close to the translocon, and there must be a system to regulate the selective glycosylation of the -Asn-X-Thr/Ser-sites. Therefore the prevailing view is that multiple subunits of the OT are required to accomplish the above requirements.
The functions of the various domains of three single transmembrane containing proteins (Ost1p, Wbp1p, and Ost4p) have been studied by site-directed mutagenesis and polyleucine replacement. Both Ost1p and Wbp1p have short cytosolic tails, and deletion of this tail has no effect on the growth phenotype of the yeast cell, indicating they are not essential for the function of Ost1p and Wbp1p (19, 20). With respect to their transmembrane domains, the specific transmembrane sequence of Ost1p is not required for its function; instead, it appears only to be important in anchoring the protein to the ER membrane (19). In contrast, the specific sequences of the transmembrane domains in Ost4p and Wbp1p play a critical role in mediating their physical association with their partners. Further mutagenesis studies revealed that the part of the transmembrane domain of Wbp1p that is close to the ER lumen (residues 398–406) plays an important role in incorporation of Wbp1p into the OT complex, whereas the other half that faces the cytoplasm (residues 407–414) does not (20). In the case of Ost4p, however, it is the segment closest to the cytosolic face of Ost4p that is crucial for its association with Stt3p and Ost3p (21), because mutations on this segment abolished their interaction and the resulting strains no longer grew at the elevated temperature (37 °C) (21). It was later shown by NMR structural analysis that this particular segment actually localizes in the second half of the transmembrane domain of Ost4p, the 
2 helix (residues 15–28) (Fig. 2) (22). The three-dimensional structure of Ost4p also revealed that this 2 helix is connected to another helix segment, the 1 helix formed by residues 10–14, via a kink of 37° between residues Phe-14 and Gly-15.
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FIG. 2.
Three-dimensional NMR structure of Ost4p deduced by NMR. a, the ensemble of the 20 lowest energy NMR structures of Ost4p after solvent refinement. b, ribbon view of the structure of Ost4p. Residues 18, 21, and 24 are expected to interact with Stt3p based on site-directed mutagenesis studies. Adapted from Zubkov et al. (22).
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Because all of the OT subunit proteins must be ER membrane-bound in yeast and in mammals to carry out the co-translational protein glycosylation, it is likely that one or more of the subunits may function to direct the whole complex to the site where it functions. Examination of the sequences of the S. cerevisiae OT subunits demonstrated that only Wbp1p was found to contain the "KKXX" motif, which is believed to function as the ER retention signal of type I membrane proteins. However, deletion of this segment did not lead to a significant growth defect in yeast cells, suggesting that it does not function to retain the OT complex in the ER (20, 23). In contrast, in the mammalian system it has been shown (24, 25) that when one of the components of OT complex was mislocated by mutations in its own retention signal, it could be retained in the ER via its interaction with other subunits. Three mammalian OT subunits, ribophorin I, ribophorin II, and OST48 (mammalian homologs of yeast Ost1p, Swp1p, and Wbp1p, respectively), were found to contain specific ER localization signals. Replacement of two Lys residues in OST48 by two serine residues (OST48ss) resulted in the mislocalization of the protein to the plasma membrane. However, if it is co-expressed with ribophorin I, ribophorin II, or chimeras that contain domains of ribophorin I, ribophorin II, and the Tac antigen (interleukin-2 receptor chain precursor), which by themselves do not exit from the ER, OST48ss could be retained in the ER. Similar observations were found with ribophorin II (24, 25). Therefore, it is possible that ribophorin I, ribophorin II, and OST48 may play a critical role in maintaining the mammalian OT complex in the ER.
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Mechanistic Complexities of OT: Diverse Functions of Certain Subunits
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In the context of the mechanistic complexities, some of the OT subunits have been found to participate in processes other than catalyzing protein N-glycosylation per se. Stt3p was recently found to be involved in biosynthesis of the cell wall 
-1,6-glucan of S. cerevisiae by means of its interaction with two KRE gene products, Kre5p and Kre9p (26). It was also proposed to interact with the protein kinase C cascade components through its N-terminal domain based on the distinctive staurosporine sensitivity caused by mutations in one of the cytosolic oriented loops near its N terminus (amino acids 158–168) (27). Moreover, Koiwa et al. (28) found that one of the two plant STT3 isoforms, STT3a in Arabidopsis, is involved in adaptive responses to salt/osmotic stress in the plant cell and has been proposed to directly or indirectly play a critical role in plant growth and/or developmental processes. A genomic scale function annotation based on the evolutionary sequence and structure similarity of proteins has implied that Ost3p and Ost6p may bear disulfide oxidoreductase activity (29). However, there is no experimental evidence so far to validate this prediction. In fact attempts to detect the oxidoreductase activity of N33, the human homolog of Ost3p/Ost6p, were unsuccessful (29).
In contrast, very limited information is available about the role of Ost2p and Ost5p. Ost5p was hypothetically proposed to associate with Ost1p based on observations that 1) it could be cross-linked with Ost1p (18) and 2) overexpression of OST5 but not overexpression of other OT subunits partially suppressed the temperature-sensitive phenotype of an ost1–5
alg5 strain (30). In the case of the essential gene product Ost2p, its human homolog, DAD1, has been shown to be required for the structural integrity of the OT complex and is also involved in apoptosis (7, 31, 32), but no possible similar function has been found in the case of Ost2p in yeast.
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Catalytic Mechanism of OT: Stt3p Is Essential Component
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In the case of OT complex, the key question of the catalytic mechanism is which of the subunits recognizes Asn-X-Thr/Ser sequences in the polypeptide substrates. In an earlier photolabeling experiment, Yan et al. (33) found that an acceptor peptide-based photoprobe, 125I-labeled Asn-Bpa-Thr-NH2, was able to bind to the luminal domain of Ost1p. As a result, Ost1p was suggested to be the subunit that is responsible for peptide substrate binding. However, after the covalent attachment site in Ost1p was narrowed down to a 9 amino acid segment (19), it was found that various mutations on this region did not cause significant defects in photolabeling or glycosylation of membrane bound and soluble glycoproteins in vivo suggesting that the luminal part of Ost1p is in close proximity to the substrate binding and/or the catalytic site, but is not directly involved in substrate recognition (19). Rather, Stt3p emerged as a strong candidate to be directly involved in the recognition/catalysis process based on subsequent photo-labeling studies in which synthetic peptides which contain several photo-reactive groups in the form of benzoylphenyllanine residues at various positions were utilized as the acceptor peptides (34). After analysis of the photocross-linking products, it was found that Ost1p, Stt3p, and Ost3p were photolabeled. Because Ost3p is not an essential gene product and mutations on the segment of Ost1p where the photo probe binds had no significant effects on the growth phenotype, the focus became Stt3p, a highly conserved protein from yeast to human that is also found in certain prokaryotes (35). Extensive mutagenesis experiments demonstrated that mutations on the luminally disposed sequence from residues 516 to 520, WWDYG, in Stt3p caused lethality of the cell and impaired the ability of glycosylatable peptide to photolabel Ost1p protein, suggesting that this segment might play a critical role in the binding or catalytic activity of OT (34).
A possible limitation of Yan and Lennarz's work (34) was the utilization of a photoprobe substrate instead of a competitive inhibitor. As is well known, enzymatic reactions take place very rapidly in vivo, and it is difficult to determine whether the photolabeling occurs precisely during the substrate-enzyme recognition event or during the process of product discharge from the enzyme. It will be especially difficult to define this point under circumstances in which a conformational change may take place when the enzyme binds one of the substrates. This, in fact, has been suggested to be the case for OT based on steady state kinetic experiments (36). Moreover, using synthetic peptide substrates instead of the nascent polypeptide that is undergoing translocation may not reflect correctly the process as it takes place in vivo. To overcome these drawbacks, Johnson and co-workers (37) performed a photocross-linking study using a growing nascent polypeptide chain that bears cryptic glycosylation sites as a substrate. In this system the potential glycosylation sites on the polypeptide were modified to QKT, a competitive inhibitor of the NXT site, and the photolabeling group was incorporated into the side chain of the Lys residue in the Lys-tRNAs. Upon photolysis, the nascent polypeptide was cross-linked only to the STT3 subunit of mammalian OT and the photocross-linking was specifically inhibited by the presence of a competitive peptide substrate of OT. This study has provided additional strong support for the idea that the active site of OT is located in Stt3p (37).
Although both of these studies (34, 37) provided strong evidence on the critical role of Stt3p in N-glycosylation, a common limitation of the experiments is that the photoreactive residue was incorporated to the X position, which can be occupied by any amino acid except for proline. Because changes at this position are allowed, even when using modified amino acids, the residue at this position must not play a critical role in enzyme-substrate recognition. This is consistent with the idea that the OT subunit which was photocross-linked by a reactive group at this position, Ost1p, is not the subunit where the binding site exists. Indeed, the NMR structural analysis (38) of a series of tripeptides that contain the Asn-X-Thr sequence and act as OT substrates in vitro has revealed that the X position is actually oriented "opposite" to the side chain of Asn and Thr, which forms an "Asn turn" structure. As shown in Fig. 3A, in a typical conformation formed by the consensus sequence, to ensure the hydroxyl group in the side chain of Thr is oriented close to the amide group in the side chain of Asn, the side chain of the X amino acid is actually oriented opposite these groups. In the case of a non-glycosylatable peptide where X is proline, as shown in Fig. 3B, the conformation of the peptide backbone is changed by the unusual rigid structure of proline. This alteration results in reorientation of the Thr residue, which led to the hydroxyl group of Thr to face away from the side chain of Asn, and the "Asn turn" is disrupted. These investigations elegantly demonstrate why proline in the X position eliminates the glycosylation of such tripeptides (38).
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FIG. 3.
Graphic representations of connectivity and hydrogen bonding information of "Asn turn" derived from spectroscopic studies of six acetyl-protected peptides. The structures shown are Ac-Asn-Ala-Thr-NH2 (a) and Ac-Asn-Pro-Thr-NH2 (b). The amide group in the side chain of Asn and the hydroxyl group in the side chain of Thr are highlighted as boldface. Adapted from Imperiali and Shannon (38).
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Despite the limitations discussed above, at present all the evidence points toward Stt3p as the subunit that plays a key role in catalyzing protein N-glycosylation by OT. In fact, there are studies suggesting that Stt3p alone is capable of recognizing and regulating the binding of both the peptide substrate and lipid-linked oligosaccharide substrate (11, 35, 36). An N-glycosylation system in which the oligosaccharide chain is structurally different from that in eukaryotic species was recently found in a prokaryotic species, Campylobacter jejuni (35). It was found that the PglB protein, a protein with strong sequence similarity to Stt3p in eukaryotic organisms, plays a critical role in catalyzing the N-glycosylation process in this prokaryotic organism and that the highly conserved amino acid motif 457WWDYGY462 located on the hydrophilic C-terminal domain of PglB oriented toward the periplasmic space is essential for the functional activity (35). These results are consistent with the earlier observation that the same motif is essential in yeast Stt3p (34). Because no other genes were found in C. jejuni with sequence similarity with the other OT subunits in eukaryotic organisms, it has been suggested the PglB protein alone bears the activity carried on by the whole OT complex in eukaryotes. Thus, it seems likely that a single protein subunit bears the ability to interact and catalyze the coupling reaction of its two substrates. In this case, however, it is very hard to understand why other essential gene products are required in higher eukaryotic organisms. Indeed, there are more studies in support of the possible role of Stt3p, which is directly involved in recognition of the other substrate in the catalytic process. Based on steady state kinetic experiments on yeast OT, Gilmore and co-workers (36) hypothetically proposed that Stt3p may be involved in regulating the recognition and binding of the dolichol-linked oligosaccharide substrate that bears different length of glycans in vitro. Recently two isoforms of OT complex were found in human cells, and the complex which contains STT3-B has been shown to be more active and display a more pronounced ability to discriminate between donor substrates with different oligosaccharide chain length than the complex containing STT3-A (11). Based on all these observations, it is reasonable to conclude that STT3 protein plays a critical role in the catalytic mechanism of OT.
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N-Glycosylation of Stt3p Itself Is Essential for Its Role in Catalyzing N-Glycosylation of Other Glycosylatable Substrates
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One interesting question about OT subunit proteins is whether they themselves are N-glycosylated. If so, does the N-glycosyl linkage play a role in glycosylation of other protein substrates? Li and co-workers (39) have investigated this question and found that only Ost1p, Wbp1p, and Stt3p are N-glycosylated proteins. Among them, the glycosylation sites of Ost1p and Wbp1p are not essential for their functions in vivo and in vitro. In contrast, mutation of two glycosylation sites (N535NT, and N539NT) of Stt3p (N535Q,T537A and N539Q,T541A) led to lethality of the cell, indicating an important function of the glycosyl linkage in Stt3p. Further mutagenesis studies as well as mass spectrometry analysis were carried out to determine whether the defect was caused by the absence of a glycan moiety or was a consequence of a conformational disruption due to the amino acid changes per se. The conclusion was that it is the glycan on the asparagine residue on the site of N539NT that is essential for the function of Stt3p.
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Amino Acids Adjacent to Consensus Sequence Help to Modulate Binding of Polypeptide Substrate with OT Complex
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Although the glycosylation site consensus sequence is a tripeptide motif of -Asn-X-Thr/Ser-, only selective -Asn-X-Thr/Ser-motifs in a glycoprotein are glycosylated (40). One possibility to explain this is that in addition to the consensus sequence, the adjacent amino acids probably also play a role in substrate-enzyme recognition. Mellquist et al. (41) examined this speculation by utilizing the rabies virus glycoprotein as substrate to study the impact of the Y amino acid in the glycosylation of Asn-X-Ser/Thr-Y. The results demonstrated that the residue at position Y can have a pronounced positive or negative effect on core glycosylation of rabies virus glycoprotein variants. Similar results were observed in the case of competitive inhibitors developed by Imperiali and co-workers (42–46). A series of peptides and peptidomimetics have been found to act as competitive inhibitors of OT. Among them, the most potent inhibitor developed was a cyclic peptidomimetic, c[Hex-Dab1-Cys2]-Thr3-Val4-Thr5-Nph6-NH2, with a Ki value of 37 nM (42, 44). A systematic study of the amino acid preference in positions 4 and 5 revealed that these residues indeed participate in modulating the binding of the inhibitor to the active site. Valine was found to be preferential over other amino acid residues at position 4, and threonine was preferred at position 5. These observations suggest that the substrate binding site on the enzyme recognizes a particular conformation encompassed by several amino acids rather than only the two specific amino acid (Asn and Ser/Thr) residues.
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Substrate Recognition and Glycopeptide Turnover at Active Site May Involve Allosteric Regulation
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It has been demonstrated that oligosaccharide chains of varying length (Glc0–2Man0–9GlcNAc2-PP-Dol) can serve as the donor substrate of OT in vivo (47) and in vitro (48). Because OT preferentially transfers the fully assembled donor substrate, Glc3Man9GlcNAc2-PP-Dol, to the nascent polypeptide chain in vivo, investigation of the kinetic differences between the various sized intermediates as substrates versus the fully assembled dolichol-linked oligosaccharide substrate would be expected to provide insight into the mechanism utilized by OT to monitor the donor substrate specificity. Trimble and co-workers (49) found that although the relative in vitro transfer rates for Glc3Man9GlcNAc2-PP-Dol and Man9GlcNAc2-PP-Dol differ by 10–25-fold, the Km values for the two donor substrates were remarkably similar, suggesting that donor substrate selection cannot simply be explained by the difference in binding affinity. Rather, a substrate activation mechanism has been proposed recently by Gilmore and co-workers based on steady-state kinetic experiments (36). Their study revealed a substantial deviation from simple Michaelis-Menten enzyme kinetics when fully assembled oligosaccharide-PP-Dol was used as the donor substrate. This deviation could be interpreted by an ordered substrate activation mechanism. That is, the fully assembled oligosaccharide donor substrate first binds to a regulatory site on the enzyme and induces a conformational change at the catalytic site. This conformational alteration in turn leads to a more favorable binding of the donor substrate at the catalytic site and further facilitates the enzymatic action (homotropic effect). It was proposed that this unique allosteric regulation mechanism may elegantly mediate the donor substrate specificity of OT (36).
The conformational regulation was also observed in the case of yeast OT in a study developed to distinguish the binding property of a peptide substrate (Bz-Asn-Leu-Thr-NHMe) and the corresponding glycopeptide product toward yeast OT (45). Compared with the binding affinity of substrate for yeast OT, N-glycosylated glycopeptide product, as expected, displayed a very low affinity for the enzyme, ensuring the efficiency of protein N-glycosylation and substrate turnover. However, glycopeptide formed by the dolichol-linked glycan and the peptide substrate mimetics in which the Asn residue was replaced by compounds such as alanine -hydrazide, alanine -hydroxylamine, and 1,3-diaminobutanoic acid displayed similar or even greater binding affinity toward OT. Conformational analysis suggested that it is the flexibility of the N-glycosyl linkage in these neoglycopeptides that allows them to adjust their configuration to be accommodated in the OT binding site, whereas the native N-glycosyl amide linkage in the normal product forms a very rigid trans conformation and is therefore immediately discharged from the binding site. Consequently it was suggested that the binding and/or catalytic site of OT is stereospecifically regulated (45). This finding, of course, adds even more potential complexities in understanding the overall process of N-glycosylation.
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Perspective
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Considerable progress has been made in unraveling the mechanism of the highly conserved protein N-glycosylation process within the past 5 years. Most importantly, Stt3p has been shown to be an essential player in a wide variety of organisms. However our understanding about the underlying mechanism is still incomplete, and several key questions remain to be determined. For example, where is the precise site on Stt3p that is responsible for peptide substrate binding? Which subunit recognizes and binds the dolichol-linked oligosaccharide donor substrate? Which subunit regulates the selective N-glycosylation of the protein? The answers to these questions will lead to our better understanding of the mechanism and regulation of oligosaccharyl transferase, which has been considered to be the "gatekeeper" to the secretory pathway (4).
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FOOTNOTES
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* This minireview will be reprinted in the 2005 Minireview Compendium, which will be available in January, 2006. Financial support for this work was provided by National Institutes of Health Grant GM33185 (to W. J. L.). 
To whom correspondence should be addressed. Tel.: 631-632-8560; Fax: 631-632-8575; E-mail: wlennarz{at}notes.cc.sunysb.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; OT, oligosaccharyl transferase.
2 A. Yan, E. Wu, and W. Lennarz, manuscript in preparation.
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ACKNOWLEDGMENTS
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We thank Dr. Robert Noiva (University of South Dakota) as well as all members of Lennarz's laboratory for critical reading of the manuscript and helpful discussion.
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TOP
INTRODUCTION
The Nine-yeast OT Subunits...