Glycobiology Advance Access originally published online on December 27, 2005
Glycobiology 2006 16(4):294-304; doi:10.1093/glycob/cwj074
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Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
1 To whom correspondence should be addressed; e-mail: suhase{at}chem.sci.osaka-u.ac.jp
Received on May 30, 2005; revised on December 15, 2005; accepted on December 26, 2005
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Abstract |
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It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-ß-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series—series 1: oligosaccharides processed from Man
1-2Man1-6 (Man1-2Man1-3)Man1-6(Man1-2Man1-2Man1-3) Manß1-4GlcNAc (M9A') and Man1-2Man1-6(Man1-3) Man1-6(Man1-2Man1-2Man1-3)Manß1-4GlcNAc (M8A') by digestion with cytosolic -mannosidase; series 2: oligosaccharides processed with Golgi -mannosidases in addition to endoplasmic reticulum (ER) and cytosolic -mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic -mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic -mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Man1-6(Man1-2Man1-2Man1-3)Manß1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic -mannosidase, and Man1-6(Man1- 2Man1-3)Manß1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic -mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi -mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol. Key words: cytosol / endoplasmic reticulum-associated degradation / free oligosaccharides / N-glycosylation / oligosaccharide sequence
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Introduction |
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The presence of cytosolic free oligosaccharides has been reported in several animal cells (Cacan et al., 1980
; Anumula and Spiro, 1983; Moore and Spiro, 1994; Kmiecik et al., 1995; Cacan and Verbert, 1999, 2000; Iwai et al., 1999; Ohashi et al., 1999; Spiro, 2004). Oligosaccharides such as (Man)4–9GlcNAc1, together with GlcMan9GlcNAc1–GlcMan5GlcNAc1, were detected in the cytosol of permeabilized HepG2 cells (Moore and Spiro, 1994), Chinese hamster ovary (CHO) cells (Kmiecik et al., 1995), calf thyroid slices (Anumula and Spiro, 1983), mouse splenocytes (Cacan et al., 1987), Madin–Darby bovine kidney cells (Cacan et al., 1996), and fibroblasts (Cacan et al., 1989). Studies on the processing of oligosaccharides using cultured cells labeled with [3H]-Man or [14C]-GlcNAc suggested various routes for their production in the cytosol (Cacan and Verbert, 1999, 2000; Spiro, 2004). First, oligosaccharides may originate from the action of oligosaccharyltransferase on the dolichyl-diphospho-Glc3 Man9GlcNAc2 present in the lumenal face of the endoplasmic reticulum (ER) (Anumula and Spiro, 1983; Spiro and Spiro, 1991). Free Glc3Man9GlcNAc2 is then hydrolyzed to Man8–9GlcNAc2 with ER -glucosidases and ER -mannosidase I, and the products are translocated to the cytosol in an adenosine triphosphate-dependent manner (Moore et al., 1995). Second, oligosaccharides on misfolded glycoproteins in the ER are released by the peptide N-glycanase (PNGase) and translocated into the cytosol or are cleaved in the cytosol by the PNGase immediately before proteasomal degradation (Cacan and Verbert, 2000; Spiro, 2004). The chitobiose residue of the oligosaccharides produced is then hydrolyzed by cytosolic endo-ß-N-acetylglucosaminidase, and oligosaccharides with a single GlcNAc residue (GN1 type) are produced (Cacan and Verbert, 1999, 2000; Spiro, 2004). (GN1-type oligosaccharides have a single GlcNAc residue at the reducing end, and GN2 have a di-N-acetylchitobiose residue at the reducing end.) GN1-type oligosaccharides are gradually processed by cytosolic -mannosidase to M5B' (Haeuw et al., 1991; Oku and Hase, 1991; Yamashiro et al., 1997; Cacan et al., 1998). M5B' is considered to be further degraded to monosaccharides in a vesicular compartment, probably the lysosome, after translocation from the cytosol (Moore and Spiro, 1994; Grard et al., 1996; Saint-pol et al., 1997). PNGase, endo-ß-N-acetylglucosaminidase (Pierce et al., 1979; Pierce et al., 1980; Lisman et al., 1985; Kato et al., 1997), and -mannosidases in the cytosol and ER (Cacan et al., 1989; Spiro and Spiro, 1991; Daniel et al., 1994; Moore et al., 1995; Kumano et al., 1996; Weng and Spiro, 1997) are believed to participate in the formation of free oligosaccharides in the cytosol.
The free oligosaccharides present in the cytosol have been mostly analyzed by means of metabolic radiolabeling of sugar chains in pulse-chase experiments (Anumula and Spiro, 1983; Cacan et al., 1987, 1989, 1996; Spiro and Spiro, 1991; Daniel et al., 1994; Moore and Spiro, 1994; Viller et al., 1994; Kmiecik et al., 1995; Moore et al., 1995; Grard et al., 1996; Weng and Spiro, 1997). However, metabolic radiolabeling is inadequate for analyzing isomeric structures and relative molar ratios of the oligosaccharides in the cytosol, as the amount of radiolabeled oligomannose changes over the incubation period. We have analyzed the amount of oligosaccharides by the pyridylamination method in this study. As previously reported, M5B' was reported to be the major free oligosaccharide (Moore and Spiro, 1994; Kmiecik et al., 1995). The presence of M2B', M4D', M5B', G1M5B', M8C', M8A', and M9' has been reported previously in the cytosolic fractions of mouse liver, hen oviduct, and CHO cells (Kmiecik et al., 1995; Ohashi et al., 1999); however, the isomeric structures of Man6GlcNAc and Man7GlcNAc have not been analyzed. Information on the isomeric structures of Man6–7GlcNAc enables us to speculate enzymes participated in the processing of oligosaccharides as those enzymes hydrolyze Man9GlcNAc1–2 and Man8GlcNAc1–2 in a specific manner (Bonay and Hughes, 1991; Haeuw et al., 1991; Bause et al., 1992; Weng and Spiro, 1993; Hamagashira et al., 1996; Lal et al., 1998; Tremblay and Herscovics, 2000). In the present study, the isomeric structures of Man6–7GlcNAc were determined for the first time. We also analyzed the effect of the cytosolic -mannosidase inhibitor swainsonine on oligosaccharides. Our new data allowed us to evaluate potential routes for the origin of free cytosolic oligosaccharides.
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Results |
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Structural analysis of cytosolic PA-oligosaccharides
The purity of the cytosolic fraction prepared from the HepG2 cells was analyzed by measuring marker enzymes: lysosomal N-acetylglucosaminidase had 5% and ER glucose-6-phosphatase had 2% of the total activity in the fraction. We, therefore, concluded that contamination by other organelles was negligible. Free oligosaccharides were prepared from the cytosolic fraction and labeled with 2-aminopyridine. The oligosaccharides were collected in the neutral fraction by diethylaminoethyl (DEAE) anion-exchange high-performance liquid chromatography (HPLC). There was no sialidase-sensitive oligosaccharide (Arthrobacter ureafaciens, Seikagaku Co., Tokyo, Japan) in the acidic fraction from the DEAE HPLC (data not shown). Pyridylamino (PA)-oligosaccharides were further separated by size-fractionation HPLC (Figure 1) and reversed-phase HPLC (Figure 2). All peaks eluted later than 15 min were collected, and each peak was digested with jack bean
-mannosidase. Twenty-one peaks were susceptible to -mannosidase and converted to M1' or G1M4'. These peaks are indicated by numerals in Figure 2. The structure of each peak was analyzed by two-dimensional HPLC system (Figures 1 and 2) by comparing its elution position with that of standard PA-sugar chains. The structures of 18 peaks were assigned; however, Peak 7-2 and Peak 8 did not correspond to any of the standard PA-sugar chains on the map. Peak 7-2 was subjected to partial acid hydrolysis study as reported previously (Makino et al., 1998), and the structures of the hydrolysates were determined by two-dimensional sugar mapping by comparing their elution positions with those of standard PA-sugar chains. The structure of Peak 7-2 was determined to be Man1-2Man1-3(Man1-3Man1-6)Manß1-4GlcNAc-PA (M5C') by building up from those hydrolysates. Peak 8 eluted at the same position as M5B' by size-fractionation HPLC and had PA-ManNAc at its reducing end, based on a reducing-end analysis (data not shown). From these results, Peak 8 was considered to be Man1-6(Man1-2Man1-2Man1-3) Manß1-4ManNAc-PA (M5B'-ManNAc). Peak 5 was estimated to be either M4A' or M4B'; however, further structural determination was not done because of the limited amount of sample available. The structures and molar amounts (uncorrected for loss during purification) of cytosolic oligosaccharides prepared from HepG2 cells are summarized in Table I.
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The cytosolic oligosaccharides capable of binding to calreticulin
Glucose-containing oligosaccharides were precisely analyzed by affinity chromatography using a calreticulin column. Eight glucosylated oligosaccharides were identified, and their structures were analyzed by two-dimensional sugar mapping (Figure 3, Table II). C-2 did not correspond to any of the standard PA-sugar chains used and was therefore analyzed further. -Glucosidase released a single glucose from C-2. Jack bean -mannosidase also took away a single mannose from C-2. The reducing-end analysis showed that C-2 had ManNAc at its reducing end (data not shown). These results indicated that the structure of C-2 was Man1-6(Glc1-3Man1-2Man1-2Man1-3)Manß1-4ManNAc-PA (Table II). G1M4' was not detected in this experiment, whereas it was found in the cytosol. This result was not unexpected, given that the binding specificity of calreticulin meant that G1M4' could not bind (Spiro et al., 1996; Vassilakos et al., 1998). Quantitative results are summarized in Table II.
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Effects of swainsonine on the cytosolic oligosaccharides
Swainsonine, which inhibits cytosolic -mannosidase (Oku and Hase, 1991), lysosomal -mannosidase, ER -mannosidase II, and Golgi -mannosidase II (Hamagashira et al., 1996; Suzuki et al., 1997; Saint-pol et al., 1999; Spiro, 2004), was used to investigate the origin of cytosolic oligosaccharides. Cytosolic oligosaccharides were isolated from HepG2 cells treated with 60 µM swainsonine for 24 h (Wako Pure Chemical Industries, Ltd., Osaka, Japan). PA-oligosaccharides were analyzed by two-dimensional HPLC mapping in the same way as described above, and the results were compared with those of nontreated cells (Table I). The amounts of M9A' and M8A' were largely increased but that of M5B' decreased.
Structure of glycans on cytosolic glycoproteins
As is summarized in Table I, there were some M7A' and M6B' in the cytosol of HepG2 cells. These oligosaccharides seemed not to be the hydrolysis products of M9A', because of incompatibility with the substrate specificity of cytosolic -mannosidase. In contrast, the Golgi -mannosidases readily produce M7A' and M6B' oligosaccharides. We considered the possibility that cytosolic glycoproteins that had dropped out of secretion pathways might be the source of these oligosaccharides. Therefore, we analyzed the structures of glycans on the glycoproteins in the cytosolic fraction. Seven PA-glycans were prepared from cytosolic glycoproteins and isolated by HPLC. Their structures were identified on the two-dimensional sugar map, and the results are shown in Figure 4 and Table I. Cytosolic protein contained M7A and M7B structures in addition to M9A and M8A structures, and M7A', M7B', M9A', and M8A' seem to be partly from cytosolic glycoprotein.
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Discussion |
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The isomeric structures and the amounts of 21 oligosaccharides present in the cytosolic fraction from intact HepG2 cells were analyzed. Oligosaccharide degradation is not thought to have occurred during the sample preparation because the cytosolic fraction was prepared below 4°C and enzymes were heat inactivated immediately after isolation of the cytosolic fraction. The present results showed the relative molar ratios of oligosaccharides present in the cytosol of intact HepG2 cells. Such data cannot be obtained from pulse-chase experiments where the relative amount of labeled oligosaccharide varies during the course of the experiment.
GN2-type oligosaccharides were not detected in the present study. However, a previous report showed that GN2-type oligosaccharides produced in the ER from Glc3Man9GlcNAc2 linked to proteins or lipids can be transported to the cytosol (Cacan and Verbert, 2000). Moreover, the GN2-type oligosaccharides GlcMan9GlcNAc2, Man9GlcNAc2, and Man8GlcNAc2 were identified in the cytosol (Moore et al., 1995; Saint-pol et al., 1997; Durrant and Moore, 2002; Chantret et al., 2003). The present results suggest that GN1-type oligosaccharides were liberated by the combined action of peptide-N-glycanase (Suzuki et al., 1997) and endo-ß-N-acetylglucosaminidase in the cytosol without releasing oligosaccharides into the medium (Kato et al., 1997). This speculation is supported by the characteristics of purified hen oviduct endo-ß-N-acetylglucosaminidase, which is very unstable in the absence of other proteins such as bovine serum albumin. In addition, the substrate specificities of the two enzymes are complementary in that cytosolic -mannosidase does not recognize the Man1-3Manß branch, whereas endo-ß-N-acetylglucosaminidase does recognize this structure (Kato et al., 1997).
In the present study, M7A', M6B', M5C', M4E', M3D', M7B', M6E', M6D', M7F', and M7E' and G1M8A', G1M8C', G1M7E', G1M7B', G1M6E', and G1M6D' were identified for the first time in the cytosol. We also analyzed the structures and estimated the relative molar ratios of the free cytosolic oligosaccharides. These data, together with those previously reported, make it possible to speculate on the pathways by which oligosaccharides are processed in the cytosol. In intact CHO cells, about 10% of the oligosaccharides transferred to nascent polypeptides from Glc3Man9GlcNAc2-P-P-Dol are translocated to the cytosol where they were hydrolyzed (Kmiecik et al., 1995). Oligosaccharides derived from G1M9A' and M9A' are arranged in two series based on the substrate specificities of the purified cytosolic -mannosidase that hydrolyzes M9A' to M5B' (Haeuw et al., 1991; Oku and Hase, 1991; Kumano et al., 1996; Yamashiro et al., 1997). When the two series are compared, isomeric structures are similar as expected from the substrate specificity of cytosolic -mannosidase; moreover, the relative molar ratios of intermediate oligosaccharides were also similar except that G1M9A' and G1M8A' were far smaller than M9A' and M8A' (Figure 5). G3M9A' and G2M9A' were not detected either in the present study or in CHO cells (our unpublished results).
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M7A', M6B', M5C', and M4D' were also detected in addition to the oligosaccharides included in the two series. These oligosaccharides lacked the Man1-2 residue linked to the Man1-3Manß branch that could not be hydrolyzed with cytosolic -mannosidase (Haeuw et al., 1991; Oku and Hase, 1991; Grard et al., 1996; Kumano et al., 1996; Yamashiro et al., 1997). M7A' and M6B' are considered to be derived from hydrolysis products formed in the Golgi -mannosidase IA and IB pathways (Lal et al., 1998). In addition to M7A' and M6B', some G1M9A', M9A', M8A', M7B', and M6B' also seem to be hydrolysis products of Golgi -mannosidases as they were present as cytosolic glycoproteins. Our data are consistent with the report that some misfolded glycoproteins are circulated through the Golgi apparatus or pre-cis-Golgi vesicles (Bause et al., 1992; Caldwell et al., 2001; Fagioli and Sitia, 2001; Vashist et al., 2001; Spiro, 2004).
The amount of M8A' exceeded that of M8C' by a factor of 
4.5, whereas that of G1M8C' was about 2.7-fold greater than G1M8A'. This comparison indicates that G1M8A' and G1M8C' might be hydrolysis products of G1M9A', on the basis that M9A' is hydrolyzed with purified cytosolic -mannosidase to M8A' and M8C' in a molar ratio of 1:2 and that cytosolic -mannosidase does not recognize the Man1-3Manß branch. Our data show that glucosylated oligosaccharides translocated from the ER to the cytosol behaved differently from nonglucosylated oligosaccharides and that the only glucosylated oligosaccharide translocated from the ER was G1M9A'.
In previous studies using the same methods as here, we found that M5B' was the most abundant oligosaccharide in the cytosolic fraction of hen oviduct (Iwai et al., 1999), mouse liver (Ohashi et al., 1999), and CHO cells (unpublished results). The data reported here are consistent with these earlier findings as the concentration of M5B' was the highest at 1.7 nmol/108 cells (loss during preparation was not considered) among oligosaccharides in HepG2 cells. The reason why the concentration of M5B' is maintained at such a high level remains to be clarified. Man5GlcNAc was the main free oligosaccharide observed in intact CHO cells after metabolic labeling for 2 h (Kmiecik et al., 1995), although Man5GlcNAc was not the most abundant oligosaccharide found in pulse-chase experiments using permeabilized HepG2 cells (Cacan and Verbert, 1999). These results suggest that glucosylated oligosaccharides and nonglucosylated oligosaccharides were differently processed in cells and indicated that G1M5B' seemed to stay longer in the cytosol than M5B'. Overall, our data agree well with a report that G1M5' was observed in a pulse-chase experiment (Moore and Spiro, 1994). G1M5B' is not thought to enter the lysosome in common with other glucosylated oligosaccharides (Moore and Spiro, 1994; Saint-pol et al., 1999; Spiro, 2004). If G1M5B' can be translocated into the ER, then the calreticulin cycle may be affected as G1M5B' might inhibit the binding between calreticulin and misfolded monoglucosylated glycoproteins. Some of the smaller oligosaccharides, such as M3D', M4D', and M5C', were also detected at high levels in the present study; M4D' has also been found in mouse liver (Ohashi et al., 1999), hen oviduct (Iwai et al., 1999), and CHO cells (our unpublished data). Judging from their isomeric structures, these oligosaccharides seem to be derived from M7A' and M6B' by digestion with cytosolic -mannosidase, indicating that they originated from glycoproteins processed in the Golgi apparatus. M3D', M4E', and G1M4E' lack the Man1-6 residue of the trimannosyl core (Table I). These oligosaccharides cannot be the hydrolysis products of cytosolic -mannosidase based on substrate specificity (Haeuw et al., 1991; Oku and Hase, 1991; Grard et al., 1996; Yamashiro et al., 1997), suggesting instead hydrolysis with an 1-6mannosidase. It is not clear whether these oligosaccharides came from the lysosome, where 1-6mannosidase is known to reside (Daniel et al., 1994; Haeuw et al., 1994; Park et al., 2005), or whether an 1-6mannosidase is also present in the cytosol.
Inhibiting cytosolic -mannosidase with swainsonine caused an increase in the amounts of several oligosaccharides (Saint-pol et al., 1997). Of these, M9A' and M8A', translocated from misfolded glycoproteins and the ER, showed the largest increase. A similar result was also found in CHO cells (our unpublished data). Our observations are consistent with the report that Man9GlcNAc and Man8GlcNAc did not decrease with time in a pulse-chase experiment, although the isomeric structures of these oligosaccharides were not analyzed (Saint-pol et al., 1997) (Figure 6).
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M8B', M8C', GM8C', M7A', M7B', M6B', and M5A' were also increased in addition to M9A' and M8A'. An increase in M7B', M6B', and M5A' was also found in CHO cells (our unpublished data). M8B', M6B', M7A', and M5A' were probably from the processing of misfolded glycoproteins with Golgi -mannosidases and/or endo--mannosidase (Lubas and Spiro, 1987) and not hydrolyzed further in the cytosol in the presence of swainsonine. This speculation is supported by the presence of the M8B structure in the microsomal esterase considered to reside in ER, and this M8B structure seems to be from G1M9A structure by digestion with Golgi glycosidases such as endo--mannosidase (Lubas and Spiro, 1987). The increase in G1M8C' and M8C' in the presence of swainsonine suggests that glycoproteins digested with Golgi -mannosidase IB were translocated to the cytosol for degradation as the activities of cytosolic -mannosidase and ER -mannosidase II are blocked by the inhibitor. The results also suggest that a part of the oligosaccharides were the products of Golgi mannosidases.
The decrease of M4D' could be explained as a consequence of the inhibition of digestion of M6B' and M7A' by cytosolic -mannosidase. Likewise, the decreases in M5B', M4E', and M6E' may be because of inhibition of digestion of M9A' and M8A' and in GM5B' because of inhibition of G1M9A' digestion. The large reduction in M5B' suggests that most M5B' were not derived from lipid-linked oligosaccharides in HepG2 cells.
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Experimental procedures |
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Preparation of standard PA-oligosaccharides
GN2-type PA-sugar chains were previously reported (Yanagida et al., 1998
) except for G3M9A. G3M9A was prepared from starfish eggs kindly donated by Prof. M. Hoshi of Keio University (Endo et al., 1987). G1M9A', M9A', M8A', M8C', M7A', M7B', M7D', M6B', M5A', and M4C' were prepared from the corresponding GN2-type PA-sugar chains by digestion with endoglycosidase-H (Seikagaku Co.) followed by pyridylamination. M7E' and M5B' were prepared by digestion of M9A' with hen oviduct -mannosidase. M6D' and M6E' were prepared by partial digestion of M7B' with jack bean -mannosidase (Seikagaku Co.). G1M8C', G1M7B', G1M6D', G1M6E', G1M5B', and G1M4E' were prepared from G1M9A' using jack bean -mannosidase. M4D', M3B', M2B', and M1' were prepared from M5B' using jack bean -mannosidase. M8B', G1M8A', and G1M7E' were prepared by partial acid hydrolysis of M9A' using trifluoroacetic acid at 100°C for 5 min. M2A', M3D', and M7F' were prepared, respectively from M3B', M5B', and M8A' by acid hydrolysis. M4A', M4B', M3A', and M3C' were prepared from M5A' by acid hydrolysis (Yanagida et al., 1998). The structures of these oligosaccharides were determined using the partial acid hydrolysis method combined with two-dimensional HPLC method as described in Partial acid hydrolysis of PA-oligosaccharides.
Preparation of cytosolic oligosaccharides and glycoproteins from HepG2 cells
Ten dishes of confluent HepG2 cells (6 x 107 cells) were harvested and lysed in 6 ml of hypotonic buffer (250 mM mannitol, 5 mM Tris–HCl, and 2 mM MgCl2, pH 7.4), followed by homogenization with a Dounce homogenizer. The homogenate underwent two centrifugations at 1000 x g for 10 min and at 105,000 x g for 1 h to isolate the cytosolic fraction as a supernatant. Lysosomal ß-N-acetylhexosaminidase (Borooas et al., 1961) and microsomal glucose-6-phosphatase (Swanson, 1954) activities were measured to examine the purity of the prepared fraction. The cytosolic fraction was heated at 100°C for 5 min to denature proteins, and then ethanol was added at a concentration of 60% to render the proteins insoluble. The precipitated proteins were collected by centrifugation at 14,600 x g for 20 min. The precipitants were washed with 60% ethanol solution and re-centrifuged. The supernatants, representing the free oligosaccharide fraction, were combined and concentrated. The free oligosaccharide fraction was purified on a Bio-Gel P-2 column (2.5 x 140 cm, Bio-Rad, Hercules, CA) using 20 mM ammonium acetate solution, pH 6.0, as an eluent and a Dowex 50W-X2 column (0.7 x 2.0 cm, H+ form, Dow Chemicals, Midland, MI) using water as an eluent. The desalted free oligosaccharide fraction was lyophilized to obtain 32 mg of material.
Preparation of PA-oligosaccharides
Three milligrams of free oligosaccharide fraction was quantitatively pyridylaminated with 30 µL of pyridylamination reagent at 90°C for 1 h followed by reduction at 80°C for 35 min with 105 µL of borane–dimethylamine reagent in accordance with a published method (Ohashi et al., 1999). After adding 300 µL aqueous ammonia to the reaction mixture, excess reagent was extracted twice with 300 µL of chloroform. The aqueous phase was concentrated, and the residue was further purified by gel filtration on a TSK-Gel Toyoperl HW40-F column (1.0 x 37 cm, Tosoh Co., Tokyo, Japan) equilibrated with 10 mM ammonium acetate buffer, pH 6.0. The fraction between the void volume and the elution positions of PA-maltose was collected and separated by a TSK-Gel DEAE-5PW anion-exchange column (Tosoh Co.). Two eluents were used: eluent A, distilled water adjusted to pH 9.0 with aqueous ammonia; and eluent B, 1 M ammonium acetate solution, pH 9.0. The column was equilibrated with eluent A at a flow rate of 1.0 mL/min. The concentration of eluent B was increased linearly from 0 to 40% over a period of 40 min. Fractions were collected according to the elution positions of PA-asialo-, monosialo-, disialo-, trisialosugar chains prepared from 1-acid glycoprotein. The fraction eluted at the PA-asialosugar chain was collected as the neutral PA-oligosaccharide fraction. The pass-through fraction was collected as the PA-oligosaccharide fraction.
High-performance liquid chromatography
Size-fractionation HPLC was carried out using a Shodex NH2P-50 column (4.6 x 50 mm, Showa Denko, Ltd., Tokyo Japan). Two eluents were used: eluent C, acetonitrile:water:acetic acid (200:800:3, v/v/v) adjusted to pH 7.0 with aqueous ammonia; and eluent D, acetonitrile:water:acetic acid (930:70:3, v/v/v) adjusted to pH 7.0 with aqueous ammonia. The column was equilibrated with eluent C:eluent D (3:97, v/v) at a flow rate of 0.6 mL/min. After injecting a sample, the concentration of eluent C was increased from 3 to 15% over 1 min, then to 55% over 44 min.
Reversed-phase HPLC was performed using a Cosmosil 5C18-P column (1.5 x 250 mm, Nacalai Tesque, Inc., Kyoto, Japan). Two elution conditions were used. Condition 1 (used for GN1-type oligosaccharides): eluent E, 0.1 M ammonium acetate buffer, pH 6.0; and eluent F, 0.1 M ammonium acetate buffer, pH 6.0 containing 1% 1-butanol. The column was equilibrated with eluent E:eluent F (95:5) at a flow rate of 0.15 mL/min. After injecting a sample, the concentration of eluent F was increased linearly from 5 to 52% over 51 min, then to 100% over 12 min. PA-sugar chains were detected by measuring fluorescence (excitation wavelength, 310 nm; emission wavelength, 380 nm). Condition 2 (used for GN2-type oligosaccharides): eluent G, 20 mM ammonium acetate buffer pH 4.0; and eluent H: 20 mM ammonium acetate buffer, pH 4.0, containing 0.5% 1-butanol. The column was equilibrated with eluent G:eluent H (85:15) at a flow rate of 0.15 mL/min. After injecting a sample, the concentration of eluent H was increased linearly from 15 to 80% over 90 min. PA-sugar chains were detected by measuring fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm).
Two-dimensional sugar mapping
The structures of the PA-sugar chains were analyzed by two-dimensional sugar mapping. The elution positions of 93 standard PA-N-linked sugar chains have already been reported, and the introduction of a reversed-phase scale made it possible to predict the elution positions even if standard PA-N-linked sugar chains were not available (Yanagida et al., 1998). PA-sugar chains were separated by reversed-phase HPLC and size-fractionation HPLC, and the elution position of each chain was compared with that of standard PA-sugar chains on the two-dimensional sugar map. Each PA-sugar chain was then digested sequentially with exoglycosidases, and the structures of the products were analyzed on the two-dimensional sugar map as reported previously (Makino et al., 1998).
Exoglycosidase digestions
A PA-oligosaccharide (10–500 pmol) was digested at 37°C for 15 h with an exoglycosidase in a total volume of 30 µL. Digestion with -mannosidase (300 mU) was carried out in 0.05 M sodium acetate buffer, pH 4.5, and with sialidase (100 mU) in 50 µL of 0.1 M sodium acetate buffer, pH 5.0. Enzyme reactions were terminated by heating at 100°C for 3 min.
Partial acid hydrolysis of PA-oligosaccharides
Partial acid hydrolysis was performed according to the method described before (Makino et al., 1998). Briefly, 200 µL of 1 M trifluoroacetic acid was added to 200 pmol of PA-oligosaccharide; one-half of the aliquot was hydrolyzed at 100°C for 5 min and the other for 30 min. The hydrolysates were lyophilized and N-acetylated. The products were analyzed by the two-dimensional sugar-mapping method.
Analysis of PA-glycans prepared from cytosolic glycoproteins
Two hundred micrograms of cytosolic glycoproteins from HepG2 cells was subjected to hydrazinolysis (100°C for 10 h). The released glycans were N-acetylated and pyridylaminated to prepare PA-glycans according to a published method (Hase, 1994). The PA-glycans were analyzed by the two-dimensional sugar-mapping method.
Reducing-end analysis of PA-oligosaccharides
Analysis of the reducing ends of PA-oligosaccharides was performed according to a published method (Suzuki et al., 1991; Hase et al., 1992). Each PA-oligosaccharide was hydrolyzed with 4 M HCl at 100°C for 8 h followed by N-acetylation. The PA-monosaccharides obtained from this procedure were separated and quantified by anion-exchange HPLC.
Preparation of a calreticulin-conjugated affinity column
Calreticulin-glutathion-S-transferase fusion protein was prepared according to a published protocol (Smith and Corcoran, 1994). Calreticulin-glutathion-S-transferase fusion protein was dissolved in phosphate-buffered saline (PBS) containing 1% Triton X-100, and the solution was stirred overnight. Glutathion-agarose (1 mL, Sigma, St. Louis, MO) was suspended in the solution of the fusion protein, and the mixture was packed in a column (0.8 x 3 cm). The calreticulin column was washed with PBS and equilibrated with 50 mM Hepes buffer, pH 7.4. PA-oligosaccharides were applied to the calreticulin column. Unbound materials were washed out with 50 mM Hepes buffer, pH 7.4, and then bound PA-oligosaccharides were eluted with 0.1 M ammonium acetate buffer, pH 4.0. PA-oligosaccharides were analyzed by the two-dimensional sugar-mapping method.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresAcknowledgmentsReferences |
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This work was supported in part by a grant-in-aid from Japan Health Science Foundation and of the 21st Century COE (Creation of Integrated EcoChemistry) and Protein 3000 programs from the Ministry of Education, Science, Sports, and Culture of Japan for S.H., and CREST, JST for S.N.
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Abbreviations |
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CHO, Chinese hamster ovary; DEAE, diethylaminoethyl; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; HPLC, high-performance liquid chromatography; PA, pyridylamino; PBS, phosphate-buffered saline; PNGase, peptide N-glycanase
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