Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking

The membrane glycoproteins Gn and Gc of Hantaan virus (HTNV) (family Bunyaviridae) are modified by N-linked glycosylation. The glycoproteins contain six potential sites for the attachment of N-linked oligosaccharides, five sites on Gn and one on Gc. The properties of the N-linked oligosaccharide chains were analyzed by treatment with endoglycosidase H, peptide:N-glycosidase F, tunicamycin, and deoxynojirimycin and were confirmed to be completely of the high-mannose type. Ten glycoprotein gene mutants were constructed by site-directed mutagenesis, including six single N glycosylation site mutants and four double-site mutants. We determined that four sites (N134, -235, -347, and -399) on Gn and the only site (N928) on Gc in their ectodomains are utilized, whereas the fifth site on Gn (N609), which faces the cytoplasm, is not glycosylated. The importance of individual N-oligosaccharide chains varied with respect to folding and intracellular transport. The oligosaccharide chain on residue N134 was found to be crucial for protein folding, whereas single mutations at the other glycosylation sites were better tolerated. Mutation at glycosylation sites N235 and N399 together resulted in Gn misfolding. The endoplasmic reticulum chaperones calnexin and calreticulin were found to be involved in HTNV glycoprotein folding. Our data demonstrate that N-linked glycosylation of HTNV glycoproteins plays important and differential roles in protein folding and intracellular trafficking.     

Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin.

The intracellular sites of biosynthesis of the structural proteins of murine hepatitis virus A59 have been analyzed using cell fractionation techniques. The nucleocapsid protein N is synthesized on free polysomes, whereas the envelope glycoproteins E1 and E2 are translated on the rough endoplasmic reticulum (RER). Glycoprotein E2 present in the RER contains N-glycosidically linked oligosaccharides of the mannose-rich type, supporting the concept that glycosylation of this protein is initiated at the co-translational level. In contrast, O-glycosylation of E1 occurs after transfer of the protein to smooth intracellular membranes. Monensin does not interfere with virus budding from the membranes of the endoplasmic reticulum, but it inhibits virus release and fusion of infected cells. The oligosaccharide side chains of E2 obtained under these conditions are resistant to endoglycosidase H and lack fucose suggesting that transport of this glycoprotein is inhibited between the trans Golgi cisternae and the cell surface. Glycoprotein E1 synthesized in the presence of monensin is completely carbohydrate-free. This observation suggests that the intracellular transport of this glycoprotein is also blocked by monensin.     

Clearance of acute-phase plasma proteins with no, high-mannose-, hybrid-, or complex type oligosaccharide side chains by the isolated perfused rat liver

The clearance of the rat acute-phase proteins alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein with no, high-mannose, hybrid or complex type oligosaccharide side chains was determined in the isolated perfused rat liver. The differently glycosylated forms of the three proteins were obtained from rat hepatocyte primary cultures treated with different inhibitors of glycosylation. The complex type forms of the three proteins were essentially not cleared by the liver during 2 h of perfusion. Unglycosylated alpha 2-macroglobulin and alpha 1-acid glycoprotein decreased in the perfusate by about 50% after 2 h; unglycosylated alpha 1-proteinase inhibitor was not taken up by the liver. The high-mannose type forms of the three proteins were nearly totally cleared. After 2 h of perfusion 10%, 45% and 30% of the hybrid type forms of alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, respectively, were cleared. The clearance rates of high-mannose and of hybrid type glycoproteins could be reduced to the rates of complex type glycoproteins by the addition of mannan to the perfusate. It is concluded that complex type glycosylation prevents the uptake of plasma glycoproteins by the liver.     

Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin.

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.     

Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor.

The leukemogenic glycoprotein (gp55) encoded by Friend spleen focus-forming virus is predominantly retained in the rough endoplasmic reticulum (RER). However, a small proportion (ca. 5%) is processed to form a derivative that occurs on plasma membranes and causes mitosis of infected erythroblasts. We have now found that gp55 folds heterogeneously in the RER to form components with different disulfide bonds and that this difference may determine their processing fates. RER gp55 consists predominantly of monomers with intrachain disulfide bonds. In contrast, the processed molecules are disulfide-bonded dimers. These dimers are extensively modified in transit to cell surfaces by conversion of four N-linked high-mannose oligosaccharides to complex derivatives and by attachment of a sialylated O-linked oligosaccharide. The plasma membrane dimers are then slowly shed into the medium by a mechanism that involves proteolytic cleavage of approximately 25 membrane-anchoring hydrophobic amino acids from the carboxyl termini of the glycoproteins. Consequently, shed molecules have shorter polypeptide chains than cell-associated gp55. We conclude that gp55 folds into different disulfide-bonded components that do not substantially isomerize, and that only one specific dimer is competent for export from the RER. Mitogenic activity of gp55 could be caused by the cell surface dimers, by the shed derivative, or by the carboxyl-terminal hydrophobic anchors that remain in the membranes after the shedding reaction.     

Correlation of glycosylation forms with position in amino acid sequence

We surveyed published reports on about 50 glycoproteins whose amino acid sequence, glycosylation sites, and type of glycosylation at a particular site have been established. We note that high-mannose substances were rarely found at the N-terminal side of a previously glycosylated complex site. There was a very definite distribution of complex sites about the N-terminal region. Furthermore, secreted glycoproteins usually contained only complex oligosaccharides whereas membrane proteins contained both types. We suggest that the position of the glycosylation site with respect to the N-terminus affects the extent of oligosaccharide processing and subsequent presentation of complex or high-mannose structures in the mature glycoprotein. This review relates glycosylation type to its position in the known sequence of given proteins and discusses these observations in light of known glycosylation processing reactions.     

Posttranslational modification and intracellular transport of mumps virus glycoproteins

Analysis of the pronase-derived glycopeptides of isolated mumps virus glycoproteins revealed the presence of both complex and high-mannose-type oligosaccharides on the HN and F1 glycoproteins, whereas only high-mannose-type glycopeptides were detected on F2. Endoglycosidase F, a newly described glycosidase that cleaves N-linked high mannose as well as complex oligosaccharides, appeared to completely cleave the oligosaccharides linked to HN and F2, whereas F1 was resistant to the enzyme. Two distinct cleavage products of F2 were observed, suggesting the presence of two oligosaccharide side chains. Tunicamycin was found to reduce the infectious virus yield and inhibit mumps virus particle formation. The two glycoproteins, HN and F, were not found in the presence of the glycosylation inhibitor. However, two new polypeptides were detected, with molecular weights of 63,000 (HNT) and 53,000 (FT), respectively, which may represent nonglycosylated forms of the glycoproteins. Synthesis of the nonglycosylated virus-coded proteins (L, NP, P, M, pI, and pII) was not affected by tunicamycin. The formation of HN oligomers and the proteolytic cleavage of the F protein were found to occur with the same kinetics. Analysis of the time course of appearance of mumps virus glycoproteins on the cell surface suggested that dimerization of HN and cleavage of F occur immediately after their exposure on the plasma membrane.     

The position of the oligosaccharide side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi

Phytohemagglutinin (PHA), the glycoprotein lectin of Phaseolus vulgaris has two types of asparagine-linked oligosaccharides per polypeptide: a high-mannose chain with the formula (Man)8-9(GlcNAc)2 on Asn12 and a modified chain with fewer mannose residues and additional fucose and xylose residues on Asn60. Glycosylation of PHA is a cotranslational process, which occurs in the endoplasmic reticulum, and newly synthesized PHA has two high-mannose chains. Transport of PHA to the protein bodies via the Golgi complex is accompanied by the modification of one of the two high-mannose chains. Why is only one chain modified, while the other remains in the high-mannose configuration? By determining the effect of digestion with various glycosidases (alpha-mannosidase, endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F) on native and denatured PHA we obtained evidence consistent with the interpretation that the accessibility of oligosaccharide chains to modifying enzymes is of major importance in determining whether a high-mannose chain becomes modified or not. The high-mannose chain of mature undenatured PHA is only partially accessible to glycosidases, while PHA obtained from the endoplasmic reticulum has one high-mannose chain, which is readily accessible to alpha-mannosidase and endoglycosidases H and F. We show that this readily accessible chain is in the same position on the polypeptide (Asn60) as the modified oligosaccharide on mature PHA. Thus, accessibility of the oligosaccharide side-chains to processing enzymes in the Golgi determines whether a particular oligosaccharide side-chain is processed or not.     

The role of N-glycosylation for the plasma clearance of rat liver secretory glycoproteins

The clearance of total rat liver secretory glycoproteins and of alpha 1-acid glycoprotein carrying no or different types of oligosaccharide side chains was studied in vivo and in the isolated perfused rat liver. In order to obtain unglycosylated or differently glycosylated forms of secreted glycoproteins, rat hepatocyte primary cultures were incubated with various inhibitors of N-glycosylation. Tunicamycin was used for the synthesis of unglycosylated (glyco)proteins, the mannosidase I inhibitor 1-deoxymannojirimycin for the synthesis of high-mannose type and the mannosidase II inhibitor swainsonine for the synthesis of hybrid-type glycoproteins. Glycoproteins carrying carbohydrate side chains of the complex type were synthesized by control hepatocytes. In vivo and in the perfused rat liver, high-mannose-type glycoproteins were cleared at the highest rate, followed by unglycosylated and hybrid-type glycoproteins. The lowest clearance rate was found for the glycoproteins with carbohydrate side chains of the complex type. For the highly glycosylated alpha 1-acid glycoprotein the differences in clearance rates were more pronounced. The following plasma half-lives were determined in vivo: complex type, 100 min; hybrid type, 15 min; unglycosylated form, 5 min; and high-mannose type less than 1 min. In the recirculating perfused liver 28% of complex-type alpha 1-acid glycoprotein, 40% of hybrid type, 47% of unglycosylated and 93% of high-mannose-type alpha 1-acid glycoprotein were removed from the perfusate within 2 h. It is concluded that N-glycosylation and processing to complex-type oligosaccharides seems to be of great importance for the circulatory life time of plasma glycoproteins.     

Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I

Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.     

Structure and processing of the mouse mammary tumor virus glycoprotein precursor pr73env

The polyprotein precursor to the envelope glycoproteins of mouse mammary tumor virus was investigated by using subcellular fractionation procedures, pactomycin mapping techniques, tunicamycin inhibition of glycosylation, and endo-beta-N-acetyl glucosaminidase H-catalyzed removal of glycosylated residues in order to characterize the biosynthesis and processing of the precursor. The results suggest that the precursor (Pr73env) is synthesized on the rough endoplasmic reticulum as a transmembrane protein, with the carboxyl terminus remaining on the cytoplasmic side. The apoprotein as an estimated molecular weight of 60,000 and acquires five core oligosaccharide units during synthesis. Cleavage of the precursor precedes the secondary glycosylation steps and therefore probably occurs before transport to the plasma membrane. However, a minor population of Pr73env containing complex oligosaccharides was also found in the plasma membrane. The order of the glycoproteins in the precursor, as determined by pactomycin mapping, in NH2-gp52-gp36-COOH.     

Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and F0 glycoproteins.

The immunoreactivity of the Sendai virus HN and F0 glycoproteins was shown to mature before reaching the final form exhibited by the native mature proteins. The maturation process differed for the two proteins. The native F0 immunoreactivity was shown to be defined cotranslationally, and the addition of high-mannose sugar residues may represent the final step in defining the maturation of immunoreactivity. On the other hand, native HN immunoreactivity was slowly fashioned during the hour after the completion of protein synthesis. Although addition of high-mannose sugar could constitute a necessary step in this slow maturation process, it was shown not to be sufficient. Processing of high-mannose sugars and HN self-association in homodimers and homotetramers were investigated as possible steps involved in the slow maturation of HN immunoreactivity. They were found not to play a significant role. On the other hand, conformational changes presumably took place during the maturation of HN immunoreactivity. Drastic immunoreactivity differences were also demonstrated between the native and denatured forms of the glycoproteins. Possible implications of these results in defining the pathways of glycoprotein synthesis are discussed.     

Topological and regulatory aspects of dolichyl phosphate mediated glycosylation of proteins

From the time of their synthesis in the rough endoplasmic reticulum until they are secreted, packaged in lysosomes, or appear as membrane components at the cell surface, the polypeptide chains of N- and O-linked glycoproteins remain associated with intracellular membranes that are components of the secretory pathway. The various co-translational and post-translational modifications of the carbohydrate moieties of glycoproteins have been shown to occur within morphologically and functionally distinct regions of this complex membrane system. However, the sugar nucleotides, which serve as precursors to the oligosaccharide moieties of these glycoproteins, are synthesized almost exclusively in the cytoplasm. These findings raise a number of questions about the mechanisms involved in the transmembrane assembly of membrane and secretory glycoproteins. In this paper these questions are reviewed and recent studies directed towards providing answers to them are summarized. In addition, information related to the possible role of dolichyl phosphate in regulating the glycosylation of proteins is presented.     

Kinetics of interactions of sendai virus envelope glycoproteins, F and HN, with endoplasmic reticulum-resident molecular chaperones, BiP, calnexin, and calreticulin

Sendai virus envelope glycoproteins, F and HN, mature during their transport through the endoplasmic reticulum (ER) and Golgi complex. To better understand their maturation processes in the ER, we investigated the time course of their interactions with three ER- resident molecular chaperones, BiP, calnexin (CNX), and calreticulin (CRT), in Sendai virus-infected HeLa cells. Pulse-chase and immunoprecipitation analyses using antibodies against each virus glycoprotein or ER chaperone revealed that F precursor interacted with CNX transiently (t(1/2)=8 min), while HN protein displayed longer and sequential interactions with BiP (t(1/2)=8 min), CNX (t(1/2)=15 min), and CRT (t(1/2)=20 min). HN interacted with the three ER chaperones not only as a monomer but also as a tetramer for several hours, suggesting mechanism(s) to undergo chaperone-mediated quality control of an assembled HN oligomer in the ER. The kinetics of dissociation of the HN-chaperone complexes exhibited a marked delay in the presence of proteasome inhibitors, suggesting that a part of HN associated with BiP, CNX, and CRT is destined to be degraded in the proteasome-dependent pathway. Further, the associations between virus glycoproteins and CNX or CRT were impaired by castanospermine, an inhibitor of ER glucosidase I and II, confirming that these interactions require monoglucosylated oligosaccharide on F(0) and HN peptides. These findings together suggest that newly synthesized F protein undergoes rapid maturation in the ER through a transient interaction with CNX, whereas HN protein requires more complex processes involving prolonged association with BiP, CNX, and CRT for its quality control in the ER.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Dissecting glycoprotein biosynthesis by the use of specific inhibitors

It is possible to interfere with different steps in the dolichol pathway of protein glycosylation and in the processing of asparagine-linked oligosaccharides. Thus some clues about the role of protein-bound carbohydrate can be obtained by comparing the biochemical fates and functions of glycosylated proteins with their non-glycosylated counterparts, or with proteins exhibiting differences in the type of oligosaccharide side chains. Cells infected with enveloped viruses are good systems for studying both aspects of protein glycosylation, since they contain a limited number of different glycoproteins, often with well-defined functions. Tunicamycin, an antibiotic, as well as several sugar analogues have been found to act as inhibitors of protein glycosylation by virtue of their anti-viral properties. They interfere with various steps in the dolichol pathway resulting in a lack of functional lipid-linked oligosaccharide precursors. Compounds that interfere with oligosaccharide trimming represent a second generation of inhibitors of glycosylation. They are glycosidase inhibitors that interfere with the processing glucosidases and mannosidases and, as a result, the conversion of high-mannose into complex-type oligosaccharides is blocked. Depending upon the compound used, glycoproteins contain glucosylated-high-mannose, high-mannose or hybrid oligosaccharide structures instead of complex ones. The biological consequences of the alterations caused by the inhibitors are manifold: increased susceptibility to proteases, improper protein processing and misfolding of polypeptide chains, loss of biological activity and alteration of the site of virus-budding, to name but a few.     

Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 and gp110.

The processing and intracellular localization of the two predominant Epstein-Barr virus glycoproteins expressed in late lytic infection were investigated. Immune light or electron microscopy of frozen fixed sections revealed that gp110 colocalized to the endoplasmic reticulum and to the nuclear membrane with the endoplasmic reticulum-resident protein, heavy-chain-binding protein (BiP), while gp350/220 accumulated in low abundance in the endoplasmic reticulum and was present in higher abundance in cytoplasmic structures presumed to be Golgi and in plasma membranes. Consistent with endoplasmic reticulum and nuclear membrane localization, the bulk of gp110 was sensitive to endoglycosidase H, indicating high-mannose, pre-Golgi, N-linked glycosylation; while consistent with Golgi and plasma membrane localization, gp350/220 was mostly resistant to endoglycosidase H because of complex N- and O-linked glycosylation. gp350/220 was as abundant in extracellular enveloped virus as in the plasma membrane but was much less abundant or undetected in internal cytoplasmic or nuclear membranes. In contrast, gp110-specific antibodies did not label extracellular or intracellular virus. These data indicate that the major antigenic components of gp110 are not incorporated into or are occluded in virions and that gp350/220 is added to virus in cytoplasmic transit through a process of de-envelopment and re-envelopment at the plasma membrane or at post-Golgi vesicles. Consistent with cytoplasmic de-envelopment and re-envelopment at the plasma membrane was the finding of some free nucleocapsids in the cytoplasm of cells with intact nuclear membranes and nucleocapsids which appeared to bud through the plasma membrane.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.