Glucose Residues as Key Determinants in the Biosynthesis and Quality Control of Glycoproteins with N-Linked Oligosaccharides*

Although glucose residues do not occur as constituents of mature N-linked oligosaccharides in eukaryotic cells, it has been appreciated for some time that they are integral components of the polymannose oligosaccharides of newly synthesized glycoproteins and their lipid-linked precursors (1). Indeed it has been shown that they play an essential role in the cotranslational transfer of a preassembled triglucosylated oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier to asparagine in Asn-XSer(Thr) sequences on the polypeptide chain (2, 3). Moreover, it has recently become apparent that the most internal of the three glucose residues, after being brought to a terminal position through the action of ER-situated glucosidases, interacts with lectin-like chaperones to mediate proper folding and/or oligomerization during protein quality control (4, 5). From these observations it has become evident that the presence of transient glucose residues on the polymannose oligosaccharides provides ideal recognition signals for crucial biological events, which have implications for a number of disease states as well as for viral replication. A rather complicated enzymatic machinery occurs in eukaryotic cells to achieve glucose attachment (6) and removal (7, 8), and this has been studied effectively with the help of mutants and inhibitors. It is the purpose of this article to provide a succinct overview of this distinctive area of glycobiology.

Although glucose residues do not occur as constituents of mature N-linked oligosaccharides in eukaryotic cells, it has been appreciated for some time that they are integral components of the polymannose oligosaccharides of newly synthesized glycoproteins and their lipid-linked precursors (1). Indeed it has been shown that they play an essential role in the cotranslational transfer of a preassembled triglucosylated oligosaccharide (Glc 3 Man 9 GlcNAc 2 ) from a dolichyl pyrophosphoryl carrier to asparagine in Asn-X-Ser(Thr) sequences on the polypeptide chain (2,3). Moreover, it has recently become apparent that the most internal of the three glucose residues, after being brought to a terminal position through the action of ER 1 -situated glucosidases, interacts with lectin-like chaperones to mediate proper folding and/or oligomerization during protein quality control (4,5). From these observations it has become evident that the presence of transient glucose residues on the polymannose oligosaccharides provides ideal recognition signals for crucial biological events, which have implications for a number of disease states as well as for viral replication. A rather complicated enzymatic machinery occurs in eukaryotic cells to achieve glucose attachment (6) and removal (7,8), and this has been studied effectively with the help of mutants and inhibitors. It is the purpose of this article to provide a succinct overview of this distinctive area of glycobiology.

Enzymes Involved in Glucose Addition
In contrast to O-linked oligosaccharides that are built up in a stepwise manner directly on the protein, the N-linked carbohydrate unit precursor 2 is assembled on a lipid carrier before being transferred en bloc to the nascent polypeptide in the ER. Whereas the biosynthesis of the lipid-linked oligosaccharide is initiated in the cytosol, the glucose additions, which represent the terminal stages of its formation, occur on the lumenal side of the ER subsequent to a "flip" of the oligosaccharide-lipid across the membrane bilayer at the Man 5 GlcNAc 2 -P-P-Dol stage (6). Dol-P-Glc that is formed from UDP-Glc through the action of a membrane-situated synthase serves as the sugar donor for three distinct glucosyltransferases (Fig. 1). These enzymes have been identified in yeast and cloned; moreover, their distinct functions have to a large extent been elucidated through studies of Saccharomyces cerevisiae mutants (6,9,10) (Table I). Similar enzymes appear to occur in most eukaryotic cells, and indeed there is evidence that three glucosyltransferases are also present in mammalian cells (3). Trypanosomes appear to be unique in being unable to synthesize Dol-P-Glc, and consequently their lipid-linked oligosaccharide remains in the Man 9 GlcNAc 2 state (11) ( Table I). The formation of a glucosylated oligosaccharide-lipid is also impaired in a rare form of the congenital disorders of glycosylation (CDG type Ic, formerly known as carbohydrate-deficient glycoprotein syndrome, type V) in which the first glucosyltransferase appears to be absent (12,13) (Table I).

Role of Triglucosyl Sequence in N-Glycosylation of Proteins
The transfer of the preformed oligosaccharide to the nascent polypeptide by oligosaccharyltransferase clearly represents a central event in the reactions in which glucose plays a role (Fig. 1). It has been well documented that this transmembrane heterooligomeric enzyme (14,15) requires the complete triglucosyl sequence for effective action (2,3) in all eukaryotes examined, with the exception of trypanosomes where Man 9 GlcNAc 2 can be transferred (11). The key role of the triglucosyl constellation is consistent with the finding that mannose-truncated glucosylated oligosaccharides (Glc 3 Man 5 GlcNAc 2 ) from a mouse lymphoma Dol-P-Man synthasedeficient mutant (16) and glucose-starved cells (17,18) can effectively be cotranslationally transferred to protein. From in vitro and in vivo experiments (19) it has been deduced that the oligosaccharyltransferase also has a hydrolytic action in which Glc 3 Man 9 GlcNAc 2 is released from its dolichyl pyrophosphate attachment when the amount of protein substrate is limiting (Fig. 1).
Because it has been shown that the ER-situated glucosidases in both intact and disrupted cells can act on triglucosylated lipidlinked oligosaccharides (Fig. 1), the possibility that they contribute to the regulation of the level of this precursor through a glucosyltransferase-glucosidase shuttle has been suggested (19).

Enzymatic Machinery Involved in Trimming Glucose
Residues from N-Linked Oligosaccharides Subsequent to the cotranslational transfer of the triglucosylated polymannose oligosaccharide to asparagine in the polypeptide chain it becomes imperative that the three ␣-linked glucose residues be released in order that further processing to the mature carbohydrate units can take place (Figs. 2 and 3). Moreover, the two outer glucose residues have to be trimmed to make possible interaction with the lectin chaperones (see below). The initial processing is effected by an ER-situated integral membrane enzyme with a lumenally oriented catalytic domain (glucosidase I) that specifically cleaves the ␣1-2-linked glucose residue; this is followed by the action of glucosidase II, which releases both of the ␣1-3linked glucose components (Table I, Fig. 4). This dual specificity of the latter enzyme has been attributed to two distinct active sites (20). Glucosidase II appears to be a soluble lumenal enzyme that although primarily located in the ER has also been noted in the intermediate compartment (21). This enzyme is a heterodimer in which the catalytic ␣-subunit appears to be associated with another polypeptide (␤-subunit) that promotes interaction with calnexin and calreticulin (22). Glucosidase II acts optimally on oligosaccharides with untrimmed mannose branches so that excision of even one mannose residue from the 6Ј-pentamannosyl branch results in a steep fall in activity (23) ( Table II).
The third enzyme involved in glucose removal is an endo-␣mannosidase, which by effecting an internal cleavage between the glucose-substituted mannose and the remainder of the 3Ј-trimannosyl branch (Fig. 4) occupies a unique place among processing glycosidases (24). Although the physiological role of this enzyme, which is predominantly located in the Golgi complex (8,24), is to act on monoglucosylated oligosaccharides with the release of Glc␣1-3Man (Table I), it also has the capacity to release Glc 2 Man and Glc 3 Man from di-and triglucosylated polymannose species, respectively (25) (Fig. 4). The endomannosidase in contrast to glucosidase II favors oligosaccharides with truncated mannose chains (Table II). Although a small portion of the endomannosidase has been observed in the intermediate compartment, it does not colocalize with the glucosidase II present at this site. 3 All three glycosidases involved in glucose excision have been cloned from mammalian sources and appear to be distinctive gene products (22,26,27), and no homology occurs among them; moreover, the endomannosidase appears unrelated to the processing mannosidases (27). Glucosidases I and II are widely distributed in eukaryotes (28), again with the exception of trypanosomes, which lack the former enzyme (11) ( Table I). In contrast, the presence of endomannosidase is primarily confined to the phylum Chordata (28). The late evolutionary appearance of this enzyme may reflect the more prominent biological role that complex oligosaccharides assume in higher organisms. The presence of an alternate deglucosylation pathway in addition to the highly conserved glucosidase I and II trimming route would ensure that no glucosylated oligosaccharides are present on the cell surface or secreted glycoproteins.

Concerted Action of Deglucosylating Enzymes
The availability of glycosidase inhibitors and mutant cell lines has facilitated an understanding of the physiological role of the enzymes involved in glucose trimming (Table I, Figs. 3 and 4). A number of agents can impose a blockade of glucosidase I and II, among which castanospermine is particularly effective (29), whereas derivatives of Glc␣1-3Man, such as Glc␣1-3(1-deoxy) mannojirimycin, are potent inhibitors of endomannosidase (30). A CHO cell line deficient in glucosidase I activity (Lec23) has been characterized (31), and a yeast mutant (gls1-1) that lacks this enzyme has also been reported (32); in both these cell lines glycoproteins with an intact triglucosyl sequence have been observed. Furthermore, a glucosidase II-deficient mouse lymphoma cell line (PHA R 2.7) (33) has proved to be a valuable asset in evaluating the consequences of glucosidase blockade (34,35). Endomannosidase has been detected in all mammalian cells examined with the exception of CHO cells (28,30) and is also not present in the CHO mutant, Lec23 (36). The absence of the enzyme in these cells provides an in vivo system in which a complete glucosidase block can be established (30,36).
Whereas the predominant deglucosylation route is believed to be mediated by the sequential action of glucosidase I and II (Fig. 2), it has become apparent that trimming of mannose residues, although primarily a Golgi function (7,37,38), commences in the ER (Fig. 3) through the action of resident ␣1,2-mannosidases I and II (38,39), which can extensively cleave the 6Ј-pentamannosyl branch of Nlinked oligosaccharides. Because glucosidase II acts very poorly on mannose-trimmed monoglucosylated oligosaccharides, these species become suitable substrates for endomannosidase (Table II). By being situated in an intracellular location distal to the ER mannosidases the endomannosidase can deglucosylate oligosaccharides that have escaped trimming by ER glucosidases (Fig. 3). It has been shown in numerous studies that in glucosidase-inhibited or deficient cells complex N-linked oligosaccharides, ranging from 10 to 70% of control, continue to be formed (40). Furthermore, it has become evident that the deglucosylation which this requires is brought about by a glucosidase-independent pathway that generates Glc 2 Man or Glc 3 Man and Man 8 -6 GlcNAc 2 N-linked oligosaccharides and appears to be operative in all mammalian cells examined except CHO cells (30,40).

Role of Monoglucosylated N-Linked Oligosaccharides in Regulating Quality Control of Glycoproteins
In recent years convincing evidence has emerged to indicate that N-linked oligosaccharides after their trimming to the monoglucosylated form are involved in quality control by interacting with the lectin-like transmembrane chaperone calnexin and its soluble lumenal homologue, calreticulin (4,5). Because these chaperones appear to play a major role in facilitating the proper folding and oligomerization of glycoproteins before they can exit from the ER, these findings have provided a major new insight into the biologically relevant signals that glucose residues can provide in regulating the intracellular destiny of proteins.
The first evidence for such a role of glucose in the fate of newly synthesized glycoproteins came from investigations which demonstrated that blockage of glucosidase I and/or glucosidase II action by inhibitors or through their absence in mutants resulted in an accelerated degradation of a variety of glycoproteins (41)(42)(43). The molecular basis of the instability of glycoproteins with untrimmed glucose sequences became apparent from studies that demonstrated that a large array of soluble and membrane glycoproteins interact with calnexin and/or calreticulin in the ER (44) through their N-linked monoglucosylated oligosaccharides (Glc 1 Man 9 GlcNAc 2 ). Binding studies moreover indicated that both calnexin (45) and calreticulin (46) exhibit a high specificity for these incompletely trimmed carbohydrate units as di-and triglucosyl polymannose oligosaccharides failed to interact with these chaperones. It was furthermore shown that even after extensive trimming of the 6Ј-pentamannosyl branch, chaperone binding activity was retained although excision of the ␣1-6 mannose residue from Glc 1 Man 5 GlcNAc 2 resulted in a complete loss of reactivity (46). Calnexin and calreticulin or their homologues have been observed in a variety of eukaryotic cells, although the latter chaperone appears to be absent in S. cerevisiae and Schizosaccharomyces pombe (Table  I) (47).
Despite the similar specificity of the two lectin chaperones in vitro, they do not necessarily interact in vivo with the same group of glycoproteins (44,47). This difference has been attributed to the fact that calnexin is membrane-bound whereas calreticulin is a soluble chaperone (44); according to this view, oligosaccharides located in the proximity of the ER membrane would interact more readily with calnexin whereas the more distant lumenally oriented carbohydrate units would preferentially bind to calreticulin.

Nature of the Lectin-Chaperone Binding Cycle Involved in Protein Quality Control
For calnexin and calreticulin to play a role in quality control, a mechanism has to be in place to monitor the formation of properly folded and assembled glycoproteins. An intriguing model to carry out this function has been proposed (5), which to a large extent is based on the properties of a UDP-Glc:glycoprotein glucosyltransferase (47) that acts as a folding sensor (Fig. 2). This ER-situated enzyme (Table I) has the ability to reglucosylate incompletely folded proteins containing Man 9 GlcNAc 2 oligosaccharide chains but acts to a substantially lesser extent on carbohydrate units with trimmed mannose chains. Although this enzyme is believed to add glucose to the same position as that occurring in the monoglucosylated oligosaccharides resulting from glucosidase II action, it differs from the glucosyltransferases involved in assembly of the lipid-linked oligosaccharide in that UDP-Glc rather than Dol-P-Glc is the sugar donor (Table I). Because the glucosyltransferase acts exclusively on denatured glycoproteins (47) it has been proposed that subsequent to disassociation of the protein-chaperone complex by glucosidase II, proteins that are still incompletely folded are reglucosylated through the action of the glycoprotein glucosyltransferase, permitting rebinding to the chaperone (5). The cyclic de-and reglucosylating is mediated by glucosidase II and the glucosyltransferase and consequently persists until proper folding has occurred, at which time the latter enzyme will no longer function.
Because it is known that glucosidase II acts poorly on trimmed 3 C. Zuber, M. J. Spiro, B. Guhl, R. G. Spiro, and J. Roth, submitted for publication.

FIG. 1. Diagrammatic representation of the enzymatic glucosylation of the oligosaccharide-lipid precursor of N-linked oligosaccha-
rides and its cotranslational transfer to asparagine residues on proteins. The scheme shows the actions of the membrane-associated Dol-P-Glc synthase (DPG-Synth) and the ER-situated glucosyltransferases (GlcTrs) that precede the N-glycosylation by oligosaccharyltransferase (OsTr). Hydrolytic actions by oligosaccharyltransferase and glucosidases I and II (Glcase), which are believed to regulate N-glycosylation precursor levels, are also shown; the released oligosaccharides can remain intravesicular or can be transported to the cytosol. The abbreviations used are: G, Glc; M, Man; and GN, GlcNAc. monoglucosylated polymannose oligosaccharides (Table II), the cyclic model would not be effective if excision of mannose residues from the 6Ј-pentamannosyl branch through the action of the ERsituated mannosidases I and II has occurred (Fig. 3). To circumvent this difficulty it has been hypothesized (46) that the folded glycoproteins still attached to the soluble chaperone, calreticulin, would be disassociated after exit from the ER through the action of endomannosidase in the Golgi compartment as this enzyme has the distinct capacity to cleave even extensively trimmed monoglucosylated oligosaccharides (Fig. 3, Table II); this scission enables the glycoprotein to undergo the further processing reactions that lead to the mature complex carbohydrate units. The finding that endomannosidase and calreticulin occur in the Golgi in similar amounts (46) is consistent with such a proposal, and indeed the colocalization of these two proteins in this compartment has been confirmed by immunocytochemical studies. 3

ER-associated Degradation of Glycoproteins
Glycoproteins that fail to undergo correct folding and/or oligomerization are retained in the ER by the lectin chaperones and are believed in many instances to undergo proteasomal degradation after Sec61-mediated retrotranslocation (48 -50). This enhanced degradation forms the basis of a number of pathological states, including cystic fibrosis and ␣ 1 -antitrypsin deficiency in which protein misfolding and ER retention play major roles (51).
This ER-associated degradation is particularly evident in experimental situations in which the formation of monoglucosylated oligosaccharides is impaired through inhibitors or mutation (41)(42)(43). Because it is believed that removal of N-linked oligosaccharides precedes proteasomal degradation (48,49), it has been postulated that the polymannose oligosaccharides that are released into the cytosol after N-glycosylation are the by-products of this degradative process (52). Indeed, N-glycanases, which have been found in both the ER (53, 54) and cytosol (55), are most likely involved in the  In the lower part of the diagram the endomannosidase is shown acting on N-linked oligosaccharides, which are resistant to glucosidase II action because of their truncation by ER mannosidases (Manase I and II). When these trimmed N-linked oligosaccharides are attached to the soluble lectin chaperone calreticulin (CRT), migration of this complex into the Golgi apparatus permits deglucosylation and dissociation of the glycoprotein-chaperone complex. In the upper portion of the scheme the glycoprotein containing triglucosylated N-linked oligosaccharides (inside box) can escape ER-associated degradation (ERAD) after folding via a nonlectin chaperone; upon entering the Golgi deglucosylation by endomannosidase can take place, thereby making possible the subsequent formation of complex Nlinked oligosaccharides even in the presence of a glucosidase block. The monosaccharides are abbreviated as in Fig. 1.   FIG. 4. Trimming enzymes (bold letters) involved in the deglucosylation of asparagine-linked oligosaccharides of glycoproteins. The inhibitors, castanospermine (CST) and Glc␣1-3(1-deoxy)mannojirimycin (GDMJ) are shown on the left. Although Glc 1 Man is the primary product of endomannosidase action, this enzyme can also cleave tri-and diglucosylated oligosaccharides with the release of Glc 3 Man and Glc 2 Man, respectively. deglycosylation although the physiological subcellular site(s) for the carbohydrate removal has not yet been clearly established. Recent studies led to the formulation of a model (56) in which proteasomes situated on the cytosolic face of the ER membrane provide part of the driving force for the retrotranslocation of glycoproteins from the ER lumen to the cytosol, and these proteasomes are postulated to act in concert with a membrane N-glycanase to facilitate the retrotranslocation. In this scheme the GlcNAc 2 -terminating released components are converted to Glc-NAc 1 -terminating species by the action of a cytosolic endo-␤-Nacetylglucosaminidase. In an alternate but not necessarily mutually exclusive model it has been proposed that both the proteasomes involved and the N-glycanase occur free in the cytosol, and accordingly deglycosylation and proteolysis occur sequentially in this compartment after retrotranslocation of the intact glycoprotein (57).

Glucosidase Inhibitors as Anti-viral Agents
It has been reported that a number of glucosidase I and II blocking agents can function as inhibitors of virus proliferation when added to cultured cells infected with either human immunodeficiency (58) or hepatitis B viruses (59). The rationale for this effect is that the retention of the outer two glucose residues of the N-linked oligosaccharides of the viral envelope glycoproteins would interfere with their association with calnexin and/or calreticulin, and this could lead to increased degradation because of improper folding or oligomerization. Indeed, it has been shown that a number of viral envelope glycoproteins including the HIV gp160 bind to calnexin and/or calreticulin (44). Because it has been shown that some viral envelope glycoproteins can be processed by endomannosidase (60), maximum effect of the glucosidase blockade would be obtained if an inhibitor of the former enzyme were also added to the infected cells.

Concluding Remarks
It is evident from the information presented in this review that glucose residues attached to polymannose-di-N-acetylchibiose oligosaccharides play an important role in the cotranslational Nglycosylation and quality control of proteins. These glucose constituents, which are only transiently present on N-linked carbohydrate units, provide a revealing example of the use to which saccharides have been put by eukaryotic cells in determining crucial biological events as well as the elaborate enzymatic machinery that has evolved to make this possible. In particular, the realization that glucose serves as a recognition signal in the quality control process opens an exciting chapter in glycobiology that has many still unexplored implications to human disease.