Glycobiology Advance Access originally published online on January 26, 2005
Glycobiology 2005 15(6):655-666; doi:10.1093/glycob/cwi046
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Glycobiology vol. 15 no. 6 © Oxford University Press 2005; all rights reserved.
N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule
2 Department of Pathology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032; 3 Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-Mizuhoku, Nagoya 467-8603, Japan; 4 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; 5 Shoshin Group, 24 Milk Street, Newburyport, MA 01950; 6 Department of Anatomy and Cell Biology, University of Pennsylvania, School of Dental Medicine, 240 South 40th Street, Philadelphia, PA 19104; and 7 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104
1 To whom correspondence should be addressed; e-mail: ss2479{at}columbia.edu
Received on December 28, 2004; revised on January 22, 2005; accepted on January 24, 2005
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Abstract |
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Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn37, Asn247, and Asn319, although Asn37 is not efficiently glycosylated. To examine N-glycan processing at Asn247 and Asn319, full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn247 sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn319 in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn247 and Asn319. However, Asn247 had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn319. Moreover, when comparing the N-glycans at Asn319 on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn247 allows additional N-glycan processing to occur at Asn319, yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined.
Key words: N-glycan processing / N-glycosylation / rabies virus glycoprotein / sequon N-glycosylation
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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N-glycosylation is a very important posttranslational protein modification that can modulate biological activity, clearance rate, solubility, protease resistance, secretion rate, and immunogenicity (Varki, 1993
). Even a highly purified preparation of a single specific glycoprotein typically consists of a set of glycosylated variants (i.e., glycoforms) that can be heterogeneous with regard to the number of sites (i.e., sequons) that are occupied with N-glycans (i.e., macroheterogeneity) and the types of glycan structures present at each sequon (i.e., microheterogeneity).
Rabies virus glycoprotein (RGP) from the Evelyn-Rokitnicki-Abelseth strain is a 505-amino-acid type I membrane glycoprotein containing a 44-amino-acid cytoplasmic tail and a 22-amino-acid transmembrane domain (Fishbein and Robinson, 1993; Wunner et al., 1988). RGP is the only surface-exposed viral coat protein; it assembles into trimers (Gaudin et al., 1992) in the endoplasmic reticulum (Langevin et al., 2002), and its ectodomain then protrudes from the lipid envelope. RGP is the viral attachment protein responsible for host cell receptor recognition (Tuffereau et al., 1998) and for low pH-induced fusion of the viral envelope with endosomal membranes (Gaudin et al., 1993). Moreover, it is the primary target of the host humoral (Cox et al., 1977; Wiktor et al., 1973) and cellular immune responses (Celis et al., 1988; Macfarlan et al., 1986). RGP can assume at least three different conformational states (Gaudin et al., 1993): the native state (N) present at the viral surface and responsible for receptor binding, the activated hydrophobic state (A) required for the interaction of RGP with its target membrane during fusion, and the fusion inactive conformation (I) (Maillard and Gaudin, 2002).
Appropriate glycosylation of RGP is important for its proper expression and function. The 439-amino-acid extracellular domain has three potential N-linked glycosylation sequons at Asn37, Asn247, and Asn319 (Anilionis et al., 1981); the latter two are efficiently core glycosylated (Shakin-Eshleman et al., 1992). Indirect immunofluorescence studies in transfected Lec1 mutant Chinese hamster ovary (CHO) cells demonstrated that Asn247 and/or Asn319 permitted high levels of surface expression; the inefficient core glycosylation at Asn37 was still sufficient to support low levels of surface expression. Deletion of all three sequons completely blocked cell surface expression of RGP (Shakin-Eshleman et al., 1992).
The presence or absence of the transmembrane and cytoplasmic domains did not affect the efficiency of core N-glycosylation at any sequon, whereas more extensive C-terminal deletions of the extracellular domain decreased the efficiency of core glycosylation at Asn319 in an in vitro cell-free transcription/translation/glycosylation system (Shakin-Eshleman et al., 1993). A soluble form of RGP containing all three sequons (i.e., RGP[wild type, WT]T434) was highly expressed and efficiently secreted by transfected CHO cells; in addition, it was appropriately antigenic and immunogenic (Wojczyk et al., 1995). Similar to full-length RGP (i.e., RGP[WT]), RGP(WT)T434 assembled into homodimers and homotrimers (Wojczyk et al., 1995). RGP(--3)T434, a soluble form of RGP, which only contains the sequon at Asn319, was also efficiently secreted by CHO cells (Wojczyk et al., 1998). Finally, a large-scale purification method was developed that enabled the purification of significant amounts of an affinity-tagged soluble form of RGP (Wojczyk et al., 1996). Given the importance of N-glycosylation for the proper folding, assembly, transport, cell surface expression, secretion, and antigenicity of glycoproteins, and given the availability of this purification method, we decided to examine the N-glycan structures on soluble, secreted forms of RGP.
The complete analysis of N-glycans bound to individual sequons is a difficult analytical task, involving several techniques, including an efficient purification procedure, isolation of specific glycopeptides, efficient release of N-glycans, and multiple glycan separation and analysis techniques. The latter can include lectin affinity chromatography, 2D proton nuclear magnetic resonance, various methods of mass spectrometry, and anion exchange, size exclusion, and reversed-phase high-performance liquid chromatography (HPLC), all performed in combination with various fragmentation methods, such as methylation analysis or exoglycosidase treatment.
The current study directly examined whether N-glycosylation of one sequon influenced glycosylation of a second sequon present in the same molecule. Initially, we studied several full-length RGP glycosylation mutants generated by site-directed mutagenesis and expressed by CHO cells; the degree of N-glycan processing at specific sequons was assessed by endoglycosidase H (Endo H) digestion. To confirm our results demonstrating differential N-glycan processing at specific sequons, we purified two soluble, secreted RGP glycosylation mutants and then isolated specific glycopeptides and analyzed their N-glycan content using a 3D N-glycan mapping technique.
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Results |
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Glycosylation of RGP glycosylation mutants expressed by stably transfected CHO cells
Previous studies demonstrated that RGP(WT) is efficiently expressed in wild-type (Burger et al., 1991
) and Lec1 CHO cells (Shakin-Eshleman et al., 1992). Various glycosylation mutants were also expressed in Lec1 cells, and the pattern of their expression and glycosylation resembles that observed in a cell-free system (Shakin-Eshleman et al., 1992). Lec1 cells are defective in processing N-glycans, arresting them as Endo H–sensitive, Man5GlcNAc2 structures (Stanley, 1983). In Lec1 cells the level of glycosylation at each sequon in RGP was independent of the presence or absence of glycosylation at other sequons in this protein.
In the current study, we examined the effect of glycosylation at one sequon on N-glycan processing at another sequon in the same protein. To this end, plasmids encoding RGP(WT), RGP(-2-), RGP(--3), and RGP(-23) (Figure 1) were cotransfected into wild-type CHO cells along with pSV2neo, as described previously (Wojczyk et al., 1996). After selection with G418, 10 colonies representing each RGP mutant were isolated and propagated. Colonies exhibiting long-term growth were screened by immunoprecipitation and sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) for RGP expression. One colony representing each glycosylation mutant was cloned by a single round of limiting dilution to isolate subclones (data not shown). Clonal cell lines expressing each of the glycosylation mutants were metabolically labeled, and recombinant forms of RGP were immunoprecipitated from detergent cell lysates using rabbit polyclonal anti-rabies virus antiserum. Labeled RGP mutants were analyzed by SDS–PAGE and quantified using a phosphorimaging technique (Figure 2). RGP(-2-) and RGP(--3) each contain only one of the sequons present in RGP(WT); RGP(-23) only has the Asn37 sequon deleted (Figure 1).
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The degree of processing of the N-glycans present in each glycosylation mutant was examined by comparing its electrophoretic mobility before (Figure 2, lanes 2, 4, 6, 8) and after (Figure 2, lanes 3, 5, 7, 9) digestion with Endo H. When RGP(-2-) was treated with Endo H (Figure 2, lane 5), 90% of the resulting protein comigrated with a non-glycosylated form of RGP(WT) (Figure 2, lane 1) produced by in vitro cell-free transcription/translation. This demonstrates that in RGP(-2-) Asn247 is efficiently glycosylated and that 90% of the N-glycans at this site are of high-mannose type. In contrast, only 35% of RGP(--3) was digested by Endo H (Figure 2, lane 7); therefore, 65% of the N-glycans at this site are of complex type. Finally, when RGP(-23) was treated with Endo H (Figure 2, lane 9), 87% of the resulting protein was Endo H–resistant; this is virtually identical to the results with RGP(WT) (Figure 2, lane 3). In addition, no singly glycosylated intermediate is seen in lanes 3 and 9 of Figure 2; this suggests that the N-glycans on an individual RGP(WT) or RGP(-23) molecule are either all high-mannose or all of complex type. In summary, wild-type CHO cells appear to differentially process the N-glycans at the Asn247 and Asn319 sequons in RGP(-2-) and RGP(–3), respectively. In addition, the presence of both sequons in RGP(-23) appears to affect their N-glycan processing, suggesting that the presence of an N-glycan at one sequon may influence processing at another sequon. However, it is also possible that these results are due to the effects of N-glycosylation on the folding, intracellular trafficking, and cell surface expression of these forms of RGP. Indeed, some of our previous studies demonstrated that when these forms of RGP reach the cell surface, they are completely Endo H–resistant (Wojczyk et al., 1995). Therefore, to investigate this issue further, we constructed soluble, secreted forms of RGP (see later discussion). In this case, the secreted forms will be mature and the N-glycans will be processed to their full extent.
Expression and purification of RGP(WT)T441His and RGP(--3)T441His
The cDNA encoding the RGP(WT)T434 termination mutant was previously modified by introducing a hexahistidyl tag (Wojczyk et al., 1996). The resulting protein, RGP(WT) T441His, contains 441 amino acids, lacks transmembrane and cytoplasmic domains, and has a 7-amino-acid long C-terminal affinity tag (Figure 1). The pRGP(-3)T441His plasmid was obtained similarly by introducing a hexahistidyl tag into the pRGP(--3)T434 termination mutant (Shakin-Eshleman et al., 1992, 1993). RGP(--3)T441His has a single glycosylation sequon at Asn319 (Figure 1).
After transfection of pRGP(--3)T441His into wild-type CHO cells, as previously described (Wojczyk et al., 1996), and selection with G418, 10 colonies were isolated, all of which exhibited long-term growth. Each colony was then screened for secretion of soluble RGP(--3)T441His. Two colonies demonstrating high levels of secretion were expanded and cloned by a single round of limiting dilution (data not shown).
To demonstrate that affinity-tagged, secreted RGP(–3) T441His assembles appropriately, chemical cross-linking was performed, as described previously (Wojczyk et al., 1996). To this end, conditioned medium from CHO cells secreting RGP(--3)T441His, which was metabolically labeled with [35S]methionine, was harvested and treated with two different concentrations of ethylene glycol bis[succinimidylsuccinate] (EGS). Following immunoprecipitation and SDS–PAGE, monomers, dimers, and trimers were observed in the presence of EGS, and only monomers were seen in the absence of this cross-linker (data not shown). This is consistent with results obtained previously for RGP(WT)T434 and RGP(WT)T441His (data not shown; Wojczyk et al., 1996). For additional verification, the conditioned medium was also immunoprecipitated with three different monoclonal antibodies (i.e., 523-11, 509-6, and 62-80-6) recognizing several nonoverlapping, conformational epitopes on RGP (Dietzschold et al., 1988; Flamand et al., 1980; Lafon et al., 1983; Prehaud et al., 1988; Seif et al., 1985; Smith et al., 1984). Each antibody was able to immunoprecipitate similar quantities of RGP(WT)T441His and RGP(--3)T441His, suggesting that these epitopes are present in an immunologically relevant conformation on both mutants (data not shown). The same method was previously used to assess the presence of conformational epitopes of multiple different RGP mutants (Wojczyk et al., 1998).
To purify preparative quantities of both RGP(WT) T441His and RGP(--3)T441His, we used a scaled-up version of a previously described purification scheme (Wojczyk et al., 1996). A Ni(II)-NTA-agarose column was first used to adsorb recombinant protein from 5 L of conditioned medium. This provided the opportunity to not only partially purify the RGP mutants but also reduce the sample volume significantly. The fractions eluted from the Ni(II)-NTA-agarose column were then loaded onto an immunoaffinity column. The bound proteins were eluted and dialyzed against distilled water. SDS–PAGE and protein staining of these samples only demonstrated the presence of purified RGP (data not shown). Western blotting using rabbit anti-rabies polyclonal antibody revealed the same results (data not shown; Wojczyk et al., 1996). This purification procedure yielded 
9 mg and 3 mg of purified RGP(WT)T441His and RGP(--3)T441His, respectively.
Trypsin digestion and glycopeptide isolation
To separate and isolate peptides containing individual N-glycosylation sites, the RGP amino acid sequence was analyzed using computer software to choose a digestion method that would provide conditions resulting in separate, individual glycopeptides containing either site 2 (i.e., Asn247) or site 3 (i.e., Asn319). Moreover, the resulting peptides needed to be sufficiently different to ensure their efficient separation by HPLC. To this end, trypsin was predicted to digest these mutated RGP proteins in 46 places, generating numerous peptides, including the following: LMDGTWVAMQTSNETK and AYTIFNK, which contain glycosylation sites 2 and 3, respectively. This prediction was verified by the analytical HPLC separation shown in Figure 3. The use of metabolic labeling to trace-label the RGP N-glycans with [3H]mannose enabled the identification of the glycopeptide-containing HPLC fractions by the use of liquid scintillation counting. Multiple semipreparative runs of the tryptic digests obtained with RGP(WT)T441His and RGP(--3)T441His were performed on the same column, following which the relevant peaks from each separation were pooled. Peptide sequencing demonstrated that for RGP(WT)T441His, fraction 28 contained the AYTIFNK glycopeptide (i.e., site 3) and fraction 34 contained the LMDGTWVAMQTSNETK glycopeptide (i.e., site 2); in addition, for RGP(--3)T441His, fraction 29 contained the AYTIFNK glycopeptide (site 3) (Figure 3). This approach was used to prepare glycopeptides for N-glycan isolation from 7 mg and 2.25 mg of RGP(WT)T441His and RGP(--3)T441His, respectively.
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N-glycan release and separation by three successive HPLC steps
The N-glycans from the three different glycopeptides (sites 2 and 3 from RGP(WT)T441His and site 3 from RGP(--3) T441His) were released by digestion with glycoamidase A and then derivatized with 2-aminopyridine. The pyridylamino (PA)-derivatized N-glycans were initially separated by anion exchange HPLC into four fractions: neutral N-glycans and mono-, di-, and tri-sialyl N-glycans (Figure 4). The relative molar compositions of these four fractions for each of the three samples are shown in Table I.
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The purified neutral, mono-, di-, and trisialylated PA-oligosaccharide fractions were individually separated by reversed phase HPLC (Figure 5). Each separated fraction was then applied to an amide column. As a result, one neutral, one mono-, one di-, and three different trisialyl N-glycans were identified.
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Structural characterization of PA-oligosaccharides using the 2D mapping technique
Structural assignment of all of the N-glycans from the RGP glycopeptides was performed by a 2D mapping technique, described previously (Takahashi et al., 1995b). The coordinates of all of the N-glycans from the RGP glycopeptides coincided on the map (± 5%) with those of known oligosaccharide standards. The resulting neutral, mono-S, di-S, tri-a, tri-b, and tri-c PA-oligosaccharides were assigned as code numbers 210.4, 1A3-210.4, 2A4-210.4, 3A2-310.18, 3A4-300.8, and 3A4-310.8, respectively (Table II). Cochromatography, before and after exoglycosidase digestion, on reversed-phase and amide columns of each of the RGP PA-oligosaccharides with the corresponding reference compounds confirmed these assignments.
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Discussion |
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Various factors affect the glycosylation of individual sequons in glycoproteins. Some influence the efficiency of core N-glycosylation, and others influence processing of N-glycans into a variety of oligosaccharide structures. Factors directly affecting core N-glycosylation efficiency include the nature of the amino acid occupying the X position in the Asn-X-Ser/Thr sequon (Kasturi et al., 1997
; Shakin-Eshleman et al., 1996), the amino acid sequence surrounding the sequon (Mellquist et al., 1998), the speed of translocation and the presence of translocation stop signals (Nilsson and von Heijne, 2000; Schroder et al., 1999), the presence of other sequons in the glycoprotein (Doyon et al., 2002), the position of the sequon relative to the NH2 terminus (Livi et al., 1991) and the COOH terminus (Nilsson and von Heijne, 2000; Shakin-Eshleman et al., 1993), the distance between the sequon and the ribosomal P-site (Whitley et al., 1996), and the presence of a glycosylphosphatidylinositol anchor (Walmsley et al., 2001; Walmsley and Hooper, 2003). N-glycan processing can be affected by some of these factors in addition to the presence of chaperones and other folding enzymes (Parodi, 2000; Trombetta and Helenius, 1998), the rate of trafficking through the secretory pathway, the accessibility of the N-glycans to various glycosidases and glycosyltransferases, and the repertoire of glycosidases and glycosyltransferases present in the cell (Roth, 2002; Trombetta and Parodi, 2003). Finally, N-glycan processing can be modified by the level of expression of a glycoprotein in a given cell, particularly if a recombinant glycoprotein is expressed at very high levels (Jenkins et al., 1996). The current study examines whether N-glycosylation at one sequon in RGP influences N-glycan processing at another sequon in the same molecule.
Our earlier studies investigated core N-glycosylation efficiency at individual sequons in RGP. Most of these studies used simplified systems based on in vitro cell-free transcription/translation/glycosylation of RGP glycosylation mutants constructed by site-directed mutagenesis. For example, in vitro transcription/translation of RGP(WT) in the presence of dog pancreatic microsomes followed by Endo H digestion confirmed that RGP(WT) was core glycosylated with high-mannose N-glycans (Shakin-Eshleman et al., 1992). These microsomes support proteolytic cleavage of the amino-terminal signal sequence and core glycosylation at N-linked glycosylation sequons; however, they do not support the type of processing required to produce hybrid and complex type N-glycans (Walter and Blobel, 1983). For example, this type of evaluation with RGP(1--) resulted in a major band consistent with an unglycosylated version of RGP and a minor species consistent with the addition of a single N-glycan, suggesting that Asn37 is inefficiently glycosylated (Shakin-Eshleman et al., 1992). All mutants containing one or two of the remaining two sequons normally present in RGP, such as RGP(-2-), RGP(--3), and RGP(-23) yielded a single major band consistent with the efficient glycosylation of both Asn247 and Asn319, respectively (Shakin-Eshleman et al., 1992). When RGP(WT) was expressed in wild-type CHO cells, it was almost completely (90%) Endo H–resistant, consistent with the presence of complex type N-glycans. When expressed in Lec1 cells (a CHO cell line defective in the processing of N-linked oligosaccharides), recombinant RGP(WT) was completely Endo H–sensitive, consistent with the presence of only high-mannose type N-glycans. Moreover, expression of RGP glycosylation mutants in Lec1 cells confirmed that Asn247 and Asn319 were efficiently core glycosylated, whereas Asn37 was not glycosylated at all (Shakin-Eshleman et al., 1992). Immunofluorescence experiments demonstrated that high levels of cell surface expression of RGP in Lec1 cells occurred only when Asn247 and/or Asn319 were present, implying that N-glycosylation was required for appropriate folding and trafficking of this protein (Shakin-Eshleman et al., 1992). In conclusion, in both systems, the efficiency of N-glycosylation at individual sequons did not depend on the presence or absence of glycosylation at other sequons in RGP. This may be due to the occurrence of core N-glycosylation during translocation of a nascent polypeptide across the endoplasmic reticulum membrane when folding is not yet complete and when all sequons are not simultaneously accessible to the oligosaccharyltransferase.
Although the in vitro cell-free transcription/translation/glycosylation system provides simplicity, rapidity of analysis, and ease of manipulation, it does not support N-glycan processing. Thus it does not allow studies of the effects of protein sequence, structure, and folding on N-glycan processing. Therefore, we used CHO cells expressing various RGP glycosylation mutants to examine the effects of N-glycosylation at one sequon on the type of N-glycan processing at another sequon. Thus cell-associated RGP(WT) expressed by wild-type CHO cells is 90% Endo H–resistant (Figure 2), whereas it was completely Endo H–sensitive when expressed in Lec1 cells (Shakin-Eshleman et al., 1992). Interestingly, when expressed in wild-type CHO cells, 90% of RGP(-2-) contains a high-mannose type N-glycan, indicating that in the absence of other sequons, the N-glycan at Asn247 is not efficiently processed. In addition, when only Asn319 was present in RGP(--3), only 65% of the N-glycans underwent processing to complex type. Finally, when both Asn247 and Asn319 were present in RGP(-23), 87% of the N-glycans were Endo H–resistant, a virtually identical result to that found with RGP(WT). These findings identify the basic similarities and differences between the results obtained using an in vitro cell-free system and a cellular expression system. Hence, the efficiency of core N-glycosylation at individual RGP sequons did not depend on the presence or absence of glycosylation at other sequons, whereas N-glycosylation of one sequon influenced N-glycan processing at another sequon on the same protein. In addition, when the sequons at either Asn247 or Asn319 are present alone in RGP, the N-glycans at these sites are processed differently.
To confirm these observations regarding site-specific N-glycan processing, additional experiments using more advanced carbohydrate analysis methods were required. Because it is very difficult to isolate full-length transmembrane forms of RGP in sufficient amounts and with the required purity, our focus turned toward constructing soluble, secreted forms of RGP. In addition, recombinant soluble forms of RGP bear a resemblance to a naturally existing soluble form of RGP, denoted Gs, which is produced in rabies virus–infected cells by proteolytic cleavage of RGP following its insertion into the cell membrane. The Gs form contains the entire extracellular RGP domain, lacks the cytoplasmic tail and most of the transmembrane domain, and retains full antigenicity (Dietzschold et al., 1983; Wunner et al., 1983). By inserting a stop codon into the extracellular domain of RGP, we previously showed that deletion of the cytoplasmic tail, transmembrane domain, and six amino acids of the extracellular domain of RGP (i.e., the T434 form) did not affect the efficiency of core N-glycosylation (Shakin-Eshleman et al., 1993). However, more extensive C-terminal deletions of the extracellular domain (i.e., the T387 and T345 forms) did diminish the efficiency of core N-glycosylation at Asn319 (Shakin-Eshleman et al., 1993); therefore, these mutants were not considered further as candidates for purification and carbohydrate analysis.
We then showed that when RGP(WT)T434 was secreted by wild-type CHO cells, it was Endo H–resistant and Peptide N-glycosidase F (PNGase F)–sensitive, proving that it did not contain high-mannose type N-glycans. In contrast, the cell-associated form of RGP(WT)T434 produced by wild-type CHO cells was completely sensitive to both endoglycosidases, demonstrating the presence of only high-mannose type N-glycans (Wojczyk et al., 1995). Furthermore, using glycosylation inhibitors, we revealed that core glycosylation of RGP(WT)T434, as well as removal of all three glucose residues, are necessary and sufficient for efficient secretion. Nonetheless, further N-glycan processing, from high-mannose to complex type N-glycans, was not required for secretion (Wojczyk et al., 1995). Similar results were obtained for cell surface expression of membrane-anchored, full-length RGP(WT) (Wojczyk et al., 1995). Additional experiments demonstrated that N-glycosylation of at least Asn319 is required for secretion of T434 forms of RGP. Because only RGP(WT)T434 and RGP(--3)T434 are secreted by CHO cells (Wojczyk et al., 1998), we were not able to analyze the oligosaccharide present on Asn247 in RGP(-2-)T434. Therefore the current study was compared the N-glycan structures at Asn247 and Asn319 in RGP(WT)T441His with the N-glycans at Asn319 in RGP(--3) T441His. The use of hexahistidyl tagged forms of RGP permitted facile purification of significant amounts of secreted, soluble forms of these proteins; in addition, these affinity-tagged forms of RGP oligomerize appropriately and are appropriately antigenic (Wojczyk et al., 1996; present study).
Analysis of the N-glycan composition at the Asn247 and Asn319 sequons of secreted forms of RGP revealed the presence of core fucosylated, bi- and triantennary complex type N-glycans at each site. However, on RGP(WT)T441His, Asn247 contains 50% as many neutral glycans, 37% more monosialylated glycans, and 24% fewer disialylated glycans when compared with the N-glycans at Asn319 present on the same molecule. Moreover, Asn319 on RGP(--3)T441His contained 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans than Asn319 on RGP (WT)T441His. In addition, no triantennary or trisialylated N-glycans were detected at Asn319 on RGP(--3)T441His. These results suggest that the presence of N-glycans at Asn247 significantly affects N-glycan processing at Asn319, leading to increased amounts of more highly sialylated bi- and triantennary complex type N-glycans at the latter site.
How N-glycosylation at one sequon influences N-glycan processing at a distant site on the same glycoprotein remains to be determined. One can speculate that the absence of N-glycans at Asn247 affects the overall structure of RGP, which modifies the exposure of Asn319 to N-glycan modification enzymes in the secretory pathway, resulting in differences in N-glycan structure. Because appropriate glycosylation is important for the proper folding, assembly, secretion, function, and antigenicity of glycoproteins, our finding has practical implications for the production of therapeutic recombinant glycoproteins. Moreover, the glycosylation variants generated in the current study will help characterize the role of site-specific N-glycosylation in the immunogenicity of RGP, and these variants may be suitable for crystallization, which would elucidate the contributions of these specific glycans to the 3D structure of RGP.
Few studies have directly addressed the influence of N-glycosylation at one sequon on N-glycan processing at another sequon in the same protein. For example, to our knowledge, this phenomenon has only been described once before: Pfeiffer et al. (1994) found that insertion of a novel sequon at Asn58 in human tissue plasminogen activator influenced N-glycan processing at Asn117 on the same protein. In contrast, insertion of a new sequon at Asn67 on this protein had no effect on N-glycan processing (Pfeiffer et al., 1994). Similarly, Ashford et al. (1993) found that N-glycosylation at one of the two sequons (i.e., Asn159 and Asn270) in recombinant, soluble rat CD4 did not influence N-glycan processing at the other sequon in the same molecule. Likewise, the addition or deletion of existing or novel sequons on the alpha-subunit of human chorionic gonadotropin had no effect on N-glycan processing at other sites on this protein (Furuhashi and Suganuma, 2003). Although the mechanism by which N-glycosylation at one sequon may or may not influence N-glycan processing at a distant sequon on the same glycoprotein remains to be determined, the availability of these RGP mutants will be useful for these types of studies.
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Materials and methods |
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Construction of RGP glycosylation and termination mutants
The RGP glycosylation mutants lack one or more glycosylation sequons (Figure 1). The termination mutants lack the transmembrane domain, the cytoplasmic domain, and six amino acids N-terminal to the transmembrane domain, which are replaced by six histidines and one glycine (Figure 1). The plasmids encoding glycosylation mutants pRGP(-2-), pRGP(--3), and pRGP(-23) were constructed by site-directed mutagenesis of pRGP(WT), as described (Shakin-Eshleman et al., 1992
). The pRGP(WT)T441His plasmid was described previously (Wojczyk et al., 1996); the pRGP(--3) T441His plasmid was constructed similarly (Wojczyk et al., 1996).
Stable transfection and cell culture
CHO cells (Pro-5) were obtained from the American Type Culture Collection (Rockville, MD). Methods for transfection, selection, evaluating RGP expression, and isolating clonal cell lines were described in detail previously (Burger et al., 1991; Shakin-Eshleman et al., 1993; Wojczyk et al., 1995).
Metabolic labeling
Dishes (100 mm) of washed, 80% confluent cells were incubated in 5 ml methionine-free complete Dulbecco’s modified Eagle’s medium, containing 50 µCi of [35S]methionine (Amersham, Arlington Heights, IL) and 2.2 µg/ml sodium bicarbonate, 10% fetal bovine serum, 2 mM L-glutamine, and 100 µg/ml penicillin and streptomycin. After 4 h at 37°C, the cells were washed with ice-cold phosphate buffered saline (PBS; 100 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.4) containing 200 µg/ml phenylmethylsulfonyl fluoride (PMSF); scraped; washed with ice-cold PBS/PMSF; resuspended in 100 µl of 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid (EDTA), pH 7.4, containing 0.5% Nonidet P-40 and 200 µg/ml PMSF; incubated on ice for 20 min; and centrifuged at 12,000 x g for 20 min at 4°C.
For purification of secreted forms of RGP, 10 150-mm dishes of washed, 80% confluent cells were incubated for 24 h in 5 ml glucose-free complete Dulbecco’s modified Eagle’s medium containing 500 µCi of [3H]mannose (NEN, Boston, MA). The conditioned medium was centrifuged at 5000 rpm for 20 min and combined with unlabeled conditioned medium for subsequent purification.
Immunoprecipitation and analysis of radiolabeled proteins
Rabbit polyclonal anti-rabies virus antiserum (Wojczyk et al., 1998) (1:100 final dilution) and 20 µl of a 50% slurry of Protein A beads were added to 100 µl of cell lysate and incubated for 3 h at 4°C. The beads were washed three times for 10 min at 4°C with 15 mM Tris, pH 7.5, 0.5 M NaCl, 5 mM EDTA, containing 1% Nonidet P-40. RGP was eluted from the beads into Endo H buffer (60 mM Na2HPO4/NaH2PO4, pH 5.5, containing 1% SDS and 200 µg/ml PMSF) or PNGase F buffer (30 mM Na2HPO4/NaH2PO4, pH 7.2, 20 mM EDTA). Samples were divided, supplemented with 4 µl Endo H (4 mU) or PNGase F (0.8 U) in the corresponding buffer, or with buffer alone, and incubated for 24 h at 37°C. An additional volume of enzyme was added after 16 h. The samples were then mixed with 5x sample buffer, boiled, and analyzed by SDS–PAGE. After electrophoresis, the gels were visualized by autoradiography or by a phosphorimaging method, as described (Wojczyk et al., 1998).
Metal-chelate affinity chromatography
For purification of soluble forms of RGP, 170 150-mm dishes of the relevant clonal cell line were plated, each containing 2.2 x 106 cells in 30 ml of complete medium. When confluent, the conditioned media were combined and centrifuged at 5000 rpm for 20 min at 4°C. The supernatant was supplemented with 3 M NaCl (1:10 v/v) and PMSF (1:100 v/v) and loaded onto a 2-ml column of Ni(II)-NTA-agarose (Qiagen, Chadsworth, CA); the condition medium recirculated for 2 days at a flow rate of 100 ml/h. The column was washed with PBS until the A280 reverted to baseline and the RGP mutant was then eluted with 100 mM imidazole in PBS.
Immunoaffinity chromatography
The hybridoma producing the 62-80-6 anti-RGP mouse monoclonal antibody was the gift of Jean Smith (Centers for Disease Control and Prevention, Atlanta, GA) (Smith et al., 1984). Monoclonal antibody purification was described previously (Wojczyk et al., 1996). An immunoaffinity column was prepared using the 62-80-6 monoclonal antibody (190 mg), 10 ml Protein G-agarose (Gibco-BRL, Grand Island, NY), and the dimethylpimelimidate cross-linker (Sigma, St. Louis, MO), as described (Wojczyk et al., 1996).
A 32-ml sample containing a soluble RGP mutant partially purified by metal-chelate affinity chromatography was diluted 1/1 (v/v) with 50 mM Tris–HCl, pH 7.5, and loaded onto the immunoaffinity column (2 ml bed volume). The sample recirculated overnight at a flow rate of 60 ml/h. The column was washed with 10 volumes of 10 mM Tris–HCl buffer, pH 7.5 and the A280 reverted to baseline. Bound protein was eluted with 100 mM glycine-HCl, pH 3.0, and 4-ml fractions were collected into tubes containing 1 ml 0.5 M Tris–HCl, pH 7.5. Protein-containing fractions were dialyzed against distilled water using a 10-kDa molecular cutoff Micro-ProDiCon membrane (Spectrum, Houston, TX). The purity of the resulting samples was analyzed by SDS–PAGE.
Western blotting
Proteins separated by 10% SDS–PAGE were transferred to nitrocellulose (Towbin et al., 1979), which was blocked with PBS containing 5% bovine serum albumin and incubated with a 1:100 dilution of rabbit polyclonal anti-rabies virus antiserum (Wojczyk et al., 1995) for 1 h at 4°C. Horseradish peroxidase–conjugated goat anti-rabbit antibody (1:1000 dilution) was the secondary antibody and the membrane was developed with 4-chloro-1-napthol (Sigma).
Trypsin digestion
Seven milligrams and 2.25 mg of RGP(WT)T441His and RGP(--3)T441His, respectively, were each evaporated to dryness, dissolved in 255 µl 6 M guanidine-HCl, 0.35 M Tris–HCl, pH 8.4, containing 2.5 mM Na2EDTA, and transferred to glass tubes. Freshly prepared 1 M dithiothreitol in distilled water (12 µl) was added; the tubes were sealed under nitrogen and incubated for 60 min at 50°C. After cooling to room temperature, the samples were carboxymethylated with 27.3 µl of freshly prepared 1 M sodium iodoacetate in distilled water at room temperature for 30 min in the dark. The reaction was halted by adding 6 µl 1 M dithiothreitol. The samples were desalted on a PD-10 column containing Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden) using 50 mM Tris–HCl, pH 8.1, containing 1 mM CaCl2. Each sample (450 µl) was digested for 2 h at 37°C using 20 µl trypsin (1 g/ml) (TPCK-treated trypsin, Worthington Biochemical, Freehold, NJ). The reaction was stopped by adding 20 µl 1 M HCl at 4°C.
HPLC
A Waters 625 liquid chromatograph system with two pumps, a tunable absorbance detector, and a Rheodyne 9125 Injector, was used. Tryptic digests (500 µl) were injected onto a 3.9 x 150 mm C18 (5 µm) 300 Å Delta Pak reversed-phase column (Millipore Waters Chromatography) at 25°C and eluted with a 0–50% gradient of acetonitrile in 0.1 M phosphate buffer, pH 2.2 (Rosner and Robbins, 1982). Samples (0.5 ml) were collected at a flow rate of 0.5 ml/min. The radioactivity in each sample was monitored by liquid scintillation counting. Fractions containing radioactivity were diluted three times with distilled water and adsorbed onto a prewashed Sep-Pak Plus C18 cartridge (Waters, Milford MA). The cartridge was then washed with 5 ml distilled water; the peptides were eluted with 50% acetonitrile and concentrated by evaporation. Aliquots of purified peptides were analyzed by peptide sequencing.
Peptide sequencing
The purified tryptic peptides were sequenced on an Applied BioSystems 494 instrument using the manufacturer’s protocols. Briefly, Biobrene coated glass fiber filters were dried under argon gas, loaded onto the reaction cartridge, and cleaned and conditioned by running several cycles of the filter precycle. Approximately 20 pmoles of sample dissolved in 0.1% trifluoroacetic acid was loaded onto the membrane, dried under argon, and loaded, and the standard protocol was commenced. After each cycle, the liberated phenylthiohydantoin amino acid derivatives were analyzed by online reversed-phase HPLC using a 0.2 x 22 cm phenylthiohydantoin-C18 column and a gradient of solvent A (3.5% tetrahydrofuran in water, which contains ABI preconcentrated buffer A solution at 20 ml/1000 ml) and solvent B (12% isopropanol in acetonitrile) at a flow rate of 325 µl/min.
Glycosidases
Glycoamidase A from sweet almonds (Takahashi, 1977), jack bean ß-galactosidase, and jack bean ß-N-acetylhexosaminidase were purchased from Seikagaku Kogyo (Tokyo). Bovine kidney 
-L-fucosidase was purchased from Boehringer Mannheim (Mannheim, Germany). The cloned (2,3)-specific sialidase from Salmonella typhimurium LT2 was purchased from Takara Shuzo (Otsu, Japan).
Reference oligosaccharides
PA derivatives of isomalto-oligosaccharides (4–20 glucose residues) and of the reference neutral oligosaccharide (code no. 210.4) were obtained from Seikagaku Kogyo. The monosialyl N-glycan (code no. 1A3-210.4), disialyl N-glycan (code no. 2A4-210.4), and trisialyl N-glycans (code no. 3A2-310.18 and 3A4-310.8) were obtained from human 5ß1 integrin (Nakagawa et al., 1996). The tri-sialyl N-glycan (code no. 3A4-300.8) was obtained by enzymatic sialylation of a neutral triantennarry oligosaccharide (code no. 300.8 from fetuin) using an (2,3)-specific trans-sialidase from Trypanosoma cruzi (Takahashi et al., 1995a).
Preparation and derivatization of RGP N-glycans
From 7 mg of purified RGP(WT)T441His (Wojczyk et al., 1996), two specific glycopeptides were obtained by trypsin digestion: the site 2 glycopeptide (LMDGTWVAMQTSNETK) containing Asn247 (denoted WS2) and the site 3 glycopeptide (AYTIFNK) containing Asn319 (WS3). In addition, the site 3 glycopeptide (denoted MS3) was obtained from 2.25 mg of purified RGP(--3)T441His. The three glycopeptides were each treated with glycoamidase A (0.3 mU for WS2 and WS3 and 0.1 mU for MS3) in 30 µl 0.5 M citrate/phosphate buffer at pH 4.0 for 16 h to release the oligosaccharides. The mixture was finally digested with 1% (w/w) of pronase. The oligosaccharides were purified by gel filtration on a Bio-Gel P-4 column (1.0 x 40 cm) and dried (Nakagawa et al., 1995). After reductive amination with 2-aminopyridine using sodium cyanoborohydride (Yamamoto et al., 1989), the PA-oligosaccharides were purified by gel filtration on a Sephadex G-15 column (1.0 x 40 cm).
Isolation and characterization of PA-oligosaccharides by three successive HPLC separations
An aliquot ( 0.5%) of each PA-oligosaccharide mixture was separated and characterized by HPLC using a LC-10A HPLC system (Shimadzu, Japan) and a previously described 3D oligosaccharide mapping technique (Takahashi et al., 1995). The PA-oligosaccharides were detected by fluorescence using excitation and emission at 320 and 400 nm, respectively. The PA-oligosaccharides were successively separated on anion exchange (TSK gel DEAE-5PW, 7.5 x 75 mm; Tosoh, Japan), reversed-phase (Shim-pack CLC-ODS; 6 x 150 mm; Shimadzu), and amide-adsorption columns (Amide-80; 4.6 x 250 mm, Tosoh). The elution conditions were described previously (Takahashi et al., 1995; Tomiya et al., 1988). The anion exchange column separated oligosaccharides based on sialic acid number. A 2D map was prepared by plotting the elution time for each peak in Glc units on the x-axis for the reversed-phase column and on the y-axis for the amide-adsorption column. After plotting the x- and y-coordinates for all PA-oligosaccharides on the 2D map, the coordinates of a given sample were compared with those of known PA-oligosaccharides. The sample PA-oligosaccharide and the relevant standard were also coinjected onto the reversed-phase and amide columns for confirmation. The sample and the relevant standard were also digested simultaneously with exoglycosidases (discussed next), and their coordinates were again compared by this approach.
Exoglycosidase digestion
Each isolated PA-oligosaccharide (50 pmol) was digested with several exoglycosidases (i.e., ß-glycosidase, ß-N-acetylhexosaminidase, and -L-fucosidase), as described (Nakagawa et al., 1996).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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We thank Dr. J. Mozdzanowski from GlaxoSmithKline, Research and Development, King of Prussia, PA for useful discussions concerning trypsin digestion.
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Abbreviations |
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CHO, Chinese hamster ovary; EDTA, ethylenediamine tetra-acetic acid; EGS, ethylene glycol bis[succinimidylsuccinate]; Endo H, endoglycosidase H; HPLC, high-performance liquid chromatography; PA, pyridylamino; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PNGase F, peptide N-glycosidase F; RGP, rabies virus glycoprotein; SDS, sodium dodecylsulfate; WT, wild type
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Anilionis, A., Wunner, W.H., and Curtis, P.J. (1981) Structure of the glycoprotein gene in rabies virus. Nature, 294, 275–278.[CrossRef][Medline]
Ashford, D.A., Alafi, C.D., Gamble, V.M., Mackay, D.J., Rademacher, T.W., Williams, P.J., Dwek, R.A., Barclay, A.N., Davis, S.J., Somoza, C. and others. (1993) Site-specific glycosylation of recombinant rat and human soluble CD4 variants expressed in Chinese hamster ovary cells. J. Biol. Chem., 268, 3260–3267.
Burger, S.R., Remaley, A.T., Danley, J.M., Moore, J., Muschel, R.J., Wunner, W.H., and Spitalnik, S.L. (1991) Stable expression of rabies virus glycoprotein in Chinese hamster ovary cells. J. Gen. Virol., 72, 359–367.
Celis, E., Karr, R.W., Dietzschold, B., Wunner, W.H., and Koprowski, H. (1988) Genetic restriction and fine specificity of human T cell clones reactive with rabies virus. J. Immunol., 141, 2721–2728.[Abstract]
Cox, J.H., Dietzschold, B., and Schneider, L.G. (1977) Rabies virus glycoprotein. II. Biological and serological characterization. Infect. Immun., 16, 754–759.
Dietzschold, B., Wiktor, T.J., Wunner, W.H., and Varrichio, A. (1983) Chemical and immunological analysis of the rabies soluble glycoprotein. Virology, 124, 330–337.[CrossRef][Web of Science][Medline]
Dietzschold, B., Rupprecht, C.E., Tollis, M., Lafon, M., Mattei, J., Wiktor, T.J., and Koprowski, H. (1988) Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Rev. Infect. Dis., 10(suppl 4), S785–798.[Medline]
Doyon, Y., Home, W., Daull, P., and LeBel, D. (2002) Effect of C-domain N-glycosylation and deletion on rat pancreatic alpha-amylase secretion and activity. Biochem. J., 362, 259–264.[CrossRef][Web of Science][Medline]
Fishbein, D.B. and Robinson, L.E. (1993) Rabies. N. Engl. J. Med., 329, 1632–1638.
Flamand, A., Wiktor, T.J., and Koprowski, H. (1980) Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. II. The glycoprotein. J. Gen. Virol., 48, 105–109.
Furuhashi, M. and Suganuma, N. (2003) Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin alpha-subunit. Endocr. J., 50, 245–253.[CrossRef][Web of Science][Medline]
Gaudin, Y., Ruigrok, R.W., Tuffereau, C., Knossow, M., and Flamand, A. (1992) Rabies virus glycoprotein is a trimer. Virology, 187, 627–632.[CrossRef][Web of Science][Medline]
Gaudin, Y., Ruigrok, R.W., Knossow, M., and Flamand, A. (1993) Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J. Virol., 67, 1365–1372.
Jenkins, N., Parekh, R.B., and James, D.C. (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat. Biotechnol., 14, 975–981.[CrossRef][Web of Science][Medline]
Kasturi, L., Chen, H., and Shakin-Eshleman, S.H. (1997) Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors. Biochem. J., 323, 415–419.[Medline]
Lafon, M., Wiktor, T.J., and Macfarlan, R.I. (1983) Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies. J. Gen. Virol., 64, 843–851.
Langevin, C., Jaaro, H., Bressanelli, S., Fainzilber, M., and Tuffereau, C. (2002) Rabies virus glycoprotein (RVG) is a trimeric ligand for the N-terminal cysteine-rich domain of the mammalian p75 neurotrophin receptor. J. Biol. Chem., 277, 37655–37662.
Livi, G.P., Lillquist, J.S., Miles, L.M., Ferrara, A., Sathe, G.M., Simon, P.L., Meyers, C.A., Gorman, J.A., and Young, P.R. (1991) Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity. J. Biol. Chem., 266, 15348–15355.
Macfarlan, R.I., Dietzschold, B., and Koprowski, H. (1986) Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain. Mol. Immunol., 23, 733–741.[CrossRef][Web of Science][Medline]
Maillard, A.P. and Gaudin, Y. (2002) Rabies virus glycoprotein can fold in two alternative, antigenically distinct conformations depending on membrane-anchor type. J. Gen. Virol., 83, 1465–1476.
Mellquist, J.L., Kasturi, L., Spitalnik, S.L. and Shakin-Eshleman, S.H. (1998) The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. Biochemistry, 37, 6833–6837.[CrossRef][Medline]
Nakagawa, H., Kawamura, Y., Kato, K., Shimada, I., Arata, Y., and Takahashi, N. (1995) Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal. Biochem., 226, 130–138.[CrossRef][Web of Science][Medline]
Nakagawa, H., Zheng, M., Hakomori, S., Tsukamoto, Y., Kawamura, Y., and Takahashi, N. (1996) Detailed oligosaccharide structures of human integrin alpha 5 beta 1 analyzed by a three-dimensional mapping technique. Eur. J. Biochem., 237, 76–85.[Web of Science][Medline]
Nilsson, I. and von Heijne, G. (2000) Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment. J. Biol. Chem., 275, 17338–17343.
Parodi, A.J. (2000) Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem. J., 348, 1–13.[Medline]
Pfeiffer, G., Strube, K.H., Schmidt, M., and Geyer, R. (1994) Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain. Eur. J. Biochem., 219, 331–348.[Web of Science][Medline]
Prehaud, C., Coulon, P., LaFay, F., Thiers, C., and Flamand, A. (1988) Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. J. Virol., 62, 1–7.
Rosner, M.R. and Robbins, P.W. (1982) Separation of glycopeptides by high performance liquid chromatography. J. Cell. Biochem., 18, 37–47.[CrossRef][Web of Science][Medline]
Roth, J. (2002) Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem. Rev., 102, 285–303.[CrossRef][Web of Science][Medline]
Schroder, K., Martoglio, B., Hofmann, M., Holscher, C., Hartmann, E., Prehn, S., Rapoport, T.A., and Dobberstein, B. (1999) Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4. EMBO J., 18, 4804–4815.[CrossRef][Web of Science][Medline]
Seif, I., Coulon, P., Rollin, P.E., and Flamand, A. (1985) Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. J. Virol., 53, 926–934.
Shakin-Eshleman, S.H., Remaley, A.T., Eshleman, J.R., Wunner, W.H., and Spitalnik, S.L. (1992) N-linked glycosylation of rabies virus glycoprotein. Individual sequons differ in their glycosylation efficiencies and influence on cell surface expression. J. Biol. Chem., 267, 10690–10698.
Shakin-Eshleman, S.H., Wunner, W.H., and Spitalnik, S.L. (1993) Efficiency of N-linked core glycosylation at asparagine-319 of rabies virus glycoprotein is altered by deletions C-terminal to the glycosylation sequon. Biochemistry, 32, 9465–9472.[CrossRef][Medline]
Shakin-Eshleman, S.H., Spitalnik, S.L., and Kasturi, L. (1996) The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. J. Biol. Chem., 271, 6363–6366.
Smith, J.S., Sumner, J.W., and Roumillat, L.F. (1984) Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus. J. Clin. Microbiol., 19, 267–272.
Stanley, P. (1983) Selection of lectin-resistant mutants of animal cells. Methods Enzymol., 96, 157–184.[Web of Science][Medline]
Takahashi, N. (1977) Demonstration of a new amidase acting on glycopeptides. Biochem. Biophys. Res. Commun., 76, 1194–1201.[CrossRef][Web of Science][Medline]
Takahashi, N., Lee, K.B., Nakagawa, H., Tsukamoto, Y., Kawamura, Y., Li, Y.T., and Lee, Y.C. (1995a) Enzymatic sialylation of N-linked oligosaccharides using an alpha-(2,3)-specific trans-sialidase from Trypanosoma cruzi: structural identification using a three-dimensional elution mapping technique. Anal. Biochem., 230, 333–342.
Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995b) Three-dimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. Anal. Biochem., 226, 139–146.[CrossRef][Web of Science][Medline]
Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. Anal. Biochem., 171, 73–90.[CrossRef][Web of Science][Medline]
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA, 76, 4350–4354.
Trombetta, E.S. and Helenius, A. (1998) Lectins as chaperones in glycoprotein folding. Curr. Opin. Struct. Biol., 8, 587–592.[CrossRef][Web of Science][Medline]
Trombetta, E.S. and Parodi, A.J. (2003) Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol., 19, 649–676.[CrossRef][Web of Science][Medline]
Tuffereau, C., Benejean, J., Blondel, D., Kieffer, B., and Flamand, A. (1998) Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus. EMBO J., 17, 7250–7259.[CrossRef][Web of Science][Medline]
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97–130.
Walmsley, A.R. and Hooper, N.M. (2003) Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein. Biochem. J., 370, 351–355.[CrossRef][Web of Science][Medline]
Walmsley, A.R., Zeng, F., and Hooper, N.M. (2001) Membrane topology influences N-glycosylation of the prion protein. EMBO J., 20, 703–712.[CrossRef][Web of Science][Medline]
Walter, P. and Blobel, G. (1983) Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol., 96, 84–93.[Web of Science][Medline]
Whitley, P., Nilsson, I.M., and von Heijne, G. (1996) A nascent secretory protein may traverse the ribosome/endoplasmic reticulum translocase complex as an extended chain. J. Biol. Chem., 271, 6241–6244.
Wiktor, T.J., Gyorgy, E., Schlumberger, D., Sokol, F., and Koprowski, H. (1973) Antigenic properties of rabies virus components. J. Immunol., 110, 269–276.
Wojczyk, B., Shakin-Eshleman, S.H., Doms, R.W., Xiang, Z.Q., Ertl, H.C., Wunner, W.H., and Spitalnik, S.L. (1995) Stable secretion of a soluble, oligomeric form of rabies virus glycoprotein: influence of N-glycan processing on secretion. Biochemistry, 34, 2599–2609.[CrossRef][Medline]
Wojczyk, B.S., Czerwinski, M., Stwora-Wojczyk, M.M., Siegel, D.L., Abrams, W.R., Wunner, W.H., and Spitalnik, S.L. (1996) Purification of a secreted form of recombinant rabies virus glycoprotein: comparison of two affinity tags. Protein Expr. Purif., 7, 183–193.[CrossRef][Web of Science][Medline]
Wojczyk, B.S., Stwora-Wojczyk, M., Shakin-Eshleman, S., Wunner, W.H., and Spitalnik, S.L. (1998) The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein. Glycobiology, 8, 121–130.
Wunner, W.H., Dietzschold, B., Curtis, P.J., and Wiktor, T.J. (1983) Rabies subunit vaccines. J. Gen. Virol., 64, 1649–1656.
Wunner, W.H., Larson, J.K., Dietzschold, B., and Smith, C.L. (1988) The molecular biology of rabies viruses. Rev. Infect. Dis., 10(suppl 4), S771–S784.[Medline]
Yamamoto, S., Hase, S., Fukuda, S., Sano, O., and Ikenaka, T. (1989) Structures of the sugar chains of interferon-gamma produced by human myelomonocyte cell line HBL-38. J. Biochem. (Tokyo), 105, 547–555.
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