Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655-0103, USA
Received on September 26, 2000; revised on December 8, 2000; accepted on December 11, 2000.
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Abstract |
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Key words: N-linked glycosylation/lipid-linked oligosaccharides/glycosyltransferases/rough endoplasmic reticulum/dolichol/alg mutants
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Introduction |
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Methods to prepare dolichol-linked oligosaccharides from tissues or cells using sequential extractions with organic solvent mixtures were first described nearly 30 years ago (Parodi et al., 1972). Modified versions of the initial extraction procedure have been extensively used to prepare radiolabeled dolichol-linked oligosaccharides from tissue slices, tissue culture cells, and fungal cultures. The predominant dolichol-linked oligosaccharide detected in extracts prepared from 3H mannose-labeled wild type cells is Glc3Man9GlcNAc2-PP-Dol (D'Souza et al., 1992
; Aebi et al., 1996
). In contrast, large-scale OS-PP-Dol isolates are much more heterogeneous and contain a preponderance of compounds that lack glucose residues (Badet and Jeanloz, 1988
; Gibbs and Coward, 1999
). For example, an OS-PP-Dol preparation isolated from calf pancreas consisted of compounds ranging between Man5GlcNAc2-PP-Dol and Glc3Man9GlcNAc2-PP-Dol, with the most abundant species being Man9GlcNAc2-PP-Dol (Badet and Jeanloz, 1988
). Complex mixtures of radiolabeled dolichol-linked oligosaccharides have been resolved by high-pressure liquid chromatography (HPLC) on a silica gel column (Wells et al., 1981
). A partial resolution of radiolabeled Man9GlcNAc2-PP-Dol from Glc3Man9GlcNAc2-PP-Dol has been achieved by anion exchange chromatography (Spiro et al., 1979b
). Digestion of 3H mannose-labeled Glc3Man9GlcNAc2-PP-Dol with different glycosidases has been used to prepare samples of five dolichol-linked oligosaccharides (D'Souza et al., 1992
). Although the latter procedure yielded Glc3Man7GlcNAc2-PP-Dol, Glc3Man5GlcNAc2-PP-Dol, Man9GlcNAc2-PP-Dol, Man7GlcNAc2-PP-Dol, and two different isomers of Man5GlcNAc2-PP-Dol, the majority of the isolated compounds do not correspond to authentic intermediates in the assembly pathway, hence they may not be optimal substrates for the glycosyltransferases. Specific dolichol-linked oligosaccharides have been prepared by organic synthesis (GlcNAc2-PP-Dol; Lee and Coward, 1992
) or by large-scale in vitro biosynthesis of the dolichol-linked oligosaccharide followed by multiple rounds of anion exchange chromatography (GlcNAc2-PP-Dol and Man5GlcNAc2-PP-Dol; Kaushal and Elbein, 1986
; Sharma et al., 1990
).
We have developed a general method for the large-scale isolation of purified dolichol-linked oligosaccharides from mammalian tissues or yeast cells. The isolation procedure was modified to reduce hydrolysis of the oligosaccharides during early steps of the procedure. We have developed a rapid HPLC procedure to resolve dolichol-linked oligosaccharides on the basis of oligosaccharide size. Purification of Man5GlcNAc2-PP-Dol from an alg3 yeast culture demonstrates the utility of this method for the preparation of pure dolichol-linked oligosaccharide assembly intermediates. Detection of the novel compound Glc3Man7GlcNAc2-PP-Dol in an OS-PP-Dol preparation isolated from
alg3 yeast cells demonstrates that the dolichol-linked oligosaccharide purification method and the OST endpoint assay described here provide powerful analytical procedures to analyze dolichol-linked oligosaccharide biosynthesis in wild-type and mutant cells.
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Results |
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The solvent extraction procedure for the large-scale isolation of dolichol-linked oligosaccharides (Kelleher et al., 1992) was modified to increase the yield of the fully assembled donor oligosaccharides and to reduce contamination of the preparation with amphipathic compounds that are derived from other intracellular organelles. To achieve these objectives, a crude microsomal membrane fraction was isolated from porcine pancreas under isotonic conditions to minimize exposure of the OS-PP-Dol compounds to cytoplasmic and lysosomal glycosidases. As described in detail in the methods section, ice-cold solvents were used for the initial solvent extractions. When the composition of the porcine pancreas OS-PP-Dol preparation was determined by HPLC as described above, more than 70% of the glycopeptide products were derived by transfer of the fully assembled dolichol-linked oligosaccharide (Figure 1C). The OST endpoint assay indicated a yield of 0.93 nMol of OS-PP-Dol per g of tissue, which is comparable to that reported previously (Badet and Jeanloz, 1988
). Notably, the modified extraction procedure did not reduce the total yield of dolichol-linked oligosaccharides, arguing strongly against the selective loss of dolichol-oligosaccharide assembly intermediates or degradation products using this procedure.
The RER lumenal enzymes glucosidase I and II have been shown to trim the glucose residues from Glc3Man9GlcNAc2-PP-Dol in vivo when protein synthesis is blocked by cellular ATP depletion (Spiro et al., 1983; Spiro and Spiro, 1991
). A portion of the crude porcine microsome preparation was incubated for 1 h at 37°C in the absence of ATP, sugar nucleotides, and glucosidase inhibitors. When the composition of the dolichol-linked oligosaccharides isolated from the preincubated microsomes was determined using the OST endpoint assay followed by HPLC analysis (Figure 1D), it was apparent that the majority of the Glc3Man9GlcNAc2-PP-Dol had been processed to Man9GlcNAc2-PP-Dol by removal of the glucose residues.
Resolution of dolichol-linked oligosaccharides by ion exchange chromatography
Radiolabeled quantities of Man9GlcNAc2-PP-Dol can be resolved from Glc3Man9GlcNAc2-PP-Dol on a DEAE-cellulose column equilibrated in CHCl3:CH3OH:H20 (10:10:3) using an increasing linear gradient of CHCl3:CH3OH:0.1 M NH4OAc (10:10:3) (Spiro et al., 1979b). To improve the resolution between different dolichol-linked oligosaccharides, the crude preparations characterized in Figures 1C and 1D were applied to a preparative (24 ml) aminopropyl silica HPLC column equilibrated in CHCl3:CH3OH:H20 (10:10:3). Dolichol-linked oligosaccharides bind to the HPLC column, and most contaminants were recovered in the flow through and wash fractions (see below). The OS-PP-Dol compounds were eluted with a 320 ml linear gradient between solvent A (CHCl3:CH3OH:H20, 10:10:3) and 40% solvent B (CHCl3:CH3OH:2 M NH4OAc, 10:10:3). A sensitive and rapid ECL-based ConA binding assay was used to detect compounds with terminal glucose or mannose residues in the column eluate fractions (Figure 2A, 2B). The fractions were also assayed using the OST endpoint assay to determine more precisely the distribution and concentration of donor substrates in the eluate fractions (Figures 2C and 2D). When the Glc3Man9GlcNAc2-PP-Dol preparation was applied to the HPLC column, the major peak of ConA binding material eluted between fractions 66 and 74 (Figure 2A). These fractions also contained the majority of the donor substrate that could be detected with the OST endpoint assay (Figure 2C). The ConA binding assay is a semiquantitative procedure used to survey the elution profile during methods development. When equal-sized aliquots of the column fraction are assayed for ConA binding activity, the ECL signal for the most concentrated samples may not fall within the linear range. We also detect a greater ConA binding signal for Glc3Man9GlcNAc2-PP-Dol than for Man5GlcNAc2-PP-Dol when equal amounts of the OS-PP-Dol are affixed to the tube, suggesting that more than two ConA molecules can bind to a branched high-mannose oligosaccharide, as suggested by previous investigators (Moothoo and Naismith, 1998
). Consequently, the ConA binding activity for the less abundant triantennarary dolichol-linked oligosaccharides (Man8GlcNAc2-PP-Dol to Glc3Man9GlcNAc2-PP-Dol) appears to be overrepresented relative to Man5GlcNAc2-PP-Dol and Glc3Man9GlcNAc2-PP-Dol in Figure 2A. As expected, multiple peaks of ConA binding activity eluted between fractions 45 and 75 when the more complex Man9GlcNAc2-PP-Dol-enriched sample was applied to the HPLC column (Figure 2B, 2D).
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Isolation of dolichol-linked oligosaccharides from alg3 yeast cultures
The asparagine-linked glycosylation (alg) mutants of Saccharyomyces cerevisiae accumulate discrete OS-PP-Dol assembly intermediates due to mutations in genes encoding the glycosyltransferases that mediate the assembly pathway (for a review see Burda and Aebi, 1999). An OS-PP-Dol preparation isolated from an alg mutant culture should be an excellent source for the facile isolation of a homogeneous assembly intermediate by preparative HPLC. To determine whether a large-scale OS-PP-Dol isolation from an alg mutant culture was feasible, we chose the well-characterized alg3 mutant (Aebi et al., 1996
). The alg3 mutant accumulates Man5GlcNAc2-PP-Dol as the major dolichol-linked oligosaccharide (Huffaker and Robbins, 1983
) due to a deficiency in Dol-P-Man:Man5GlcNAc2-PP-Dol
1,3 mannosyltransferase activity (Verostek et al., 1991
). A subsequent structural analysis of N-linked oligosaccharides isolated from an alg3 mutant revealed that a substantial proportion of the protein-linked oligosaccharide is Glc3Man5GlcNAc2, indicating that the alg3 strain also synthesizes the glucosylated dolichol-linked oligosaccharide Glc3Man5GlcNAc2-PP-Dol (Verostek et al., 1993
). Here, we used a
alg3 mutant created by gene disruption (Aebi et al., 1996
) to eliminate traces of the fully assembled dolichol-linked oligosaccharide that can arise using the original alg3 isolate (Verostek et al., 1991
).
Dolichol-linked oligosaccharides were isolated from a 10.5-L culture of alg3 yeast that was grown to a density of 2.2 (A600). An OST endpoint analysis indicated a yield of 42 nMol of OS-PP-Dol from 72 g (wet weight) of
alg3 yeast cells. The glycopeptide products from the OST endpoint assays were analyzed by HPLC to determine which donor substrates were present (Figure 4A). The three major glycopeptide peaks comigrated with the Hex5GlcNAc2-NYT, Hex8GlcNAc2-NYT, and Hex10GlcNAc2-NYT standards. These three compounds respectively account for 71%, 15%, and 6% of the glycopeptide products. Minor peaks with 4, 6, 7, and 9 hexose units were also detected. Thus, without further purification by HPLC, an OS-PP-Dol preparation isolated from
alg3 cells contains a number of different assembly intermediates. The elution position and relative abundance of the two major glycopeptide products (Hex5GlcNAc2-NYT and Hex8GlcNAc2-NYT) is consistent with their identification as Man5GlcNAc2-NYT and Glc3Man5GlcNAc2-NYT. The percentage of each oligosaccharide donor that was utilized during a 30-min OST assay was determined by HPLC analysis (Figure 4B). Although roughly 20% of the dolichol-linked oligosaccharides had been utilized as substrates by the 30-min time point, the initial transfer rate for the Hex8GlcNAc2-PP-Dol and Hex10GlcNAc2-PP-Dol donors was more rapid. As triglucosylated dolichol-linked oligosaccharides (e.g., Glc3Man5GlcNAc2-PP-Dol or Glc3Man9GlcNAc2-Dol) are the preferred donors for the OST (Turco et al., 1977
; Spiro et al., 1979a
; Verostek et al., 1993
), the most reasonable interpretation of our results is that the novel Hex10GlcNAc2-NYT product is the triglucosylated compound Glc3Man7GlcNAc2-NYT that would arise by the addition of the
-1,6 dimannose antenna and terminal glucose residues to Man5GlcNAc2-PP-Dol. The glycopeptide products obtained from the OST endpoint assay of the
alg3 OS-PP-Dol preparation were digested with Endoglycosidase H to provide structural evidence to support this conclusion. Aliquots (25 pmol) of the
alg3 glycopeptides or a Glc3Man9GlcNAc2-NYT standard were digested for 24 h with a large excess of Endo H (500 NEB units). The digestion products were applied to the analytical HPLC column to resolve GlcNAc-NYT from any undigested glycopeptides. Unlike Glc3Man9GlcNAc2-NYT (Hex12-NYT) which was quantitatively converted to GlcNAc-NYT by the digestion, Man5GlcNAc2-NYT and Glc3Man5GlcNAc2-NYT were much less sensitive to Endo H digestion (Figure 4C) consistent with the absence of both the
1-3 and
1-6 dimannose antenna that confer sensitivity to Endo H (Maley et al., 1989
). The Hex10GlcNAc2-NYT product showed an intermediate sensitivity to Endo H digestion (Figure 4C) consistent with the presence of the
1-6 dimannose antenna.
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Analysis of OS-PP-Dol preparations by TLC on silica gel plates
The dolichol-linked oligosaccharide preparations isolated from porcine pancreas and alg3 yeast cultures were spotted onto silica gel thin layer plates and subjected to chromatography in CHCl3:CH3OH:H20 (10:10:3). Organic compounds that were resolved on the TLC plate were visualized with iodine vapor (not shown) or with an anisaldehydesulfuric acid spray reagent (Figure 5A). The OS-PP-Dol preparation isolated from the
alg3 yeast microsomes contains at least seven compounds that can be detected by staining with the anisaldehydesulfuric acid spray (Figure 5A, L) and iodine vapor (not shown). The number of compounds that could be detected on the TLC was greatest with whole tissues, intermediate with crude microsomes, and least when purified RER were used as the starting material for the OS-PP-Dol isolation (data not shown). The majority of the components in the crude OS-PP-Dol preparation did not bind to the preparative aminopropyl silica column, but were instead recovered in a flow-through fraction (Figure 5A, UB) or in the wash fractions (not shown). The TLC lanes designated as P1, P2, and P3 correspond to the three peak fractions from the column shown in Figure 4D. Similar amounts (
50 pmol) of dolichol-linked oligosaccharide were spotted onto each of the TLC lanes except for the UB fraction, which lacked detectable OS-PP-Dol. Faint spots corresponding to Man5GlcNAc2-PP-Dol (P1), Glc3Man5GlcNAc2-PP-Dol (P2), and Glc3Man7GlcNAc2-PP-Dol (P3) were visible in the region of the TLC plate designated by the bracket labeled OS-PP-Dol. More important, the major compounds visible in the crude OS-PP-Dol isolate were absent or were present in greatly reduced amounts (see solvent front in Figure 5A). As reported previously for this TLC system (Badet and Jeanloz, 1988
), the mobility of the OS-PP-Dol decreases with increasing saccharide units. The column load fraction (L) but not the flow-through fraction (UB) contains a spot that comigrates with the Man5GlcNAc2-PP-Dol spot in the P2 fraction. Additional evidence concerning the mobility of OS-PP-Dol compounds on the TLC system was obtained by probing a TLC plate with ConA-FITC (Figure 5B). The Man9GlcNAc2-PP-Dol (M9), Glc2Man9GlcNAc2-PP-Dol (G2), and Glc3Man9GlcNAc2-PP-Dol (G3) pools obtained from the preparative HPLC column (Figure 2) were resolved on TLC plates as in Figure 5A. Each of the purified OS-PP-Dol samples migrated as a single spot in the region of the plate designated by the labeled bracket. For unknown reasons, the ConA-FITC detection procedure for locating OS-PP-Dol on TLC plates was less sensitive than anticipated and showed considerable variation between experiments.
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Discussion |
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We have used a sensitive OST endpoint assay to monitor the elution of dolichol-linked oligosaccharides from the preparative HPLC column. The validity of using the OST endpoint assay to determine the concentration of OS-PP-Dol samples was verified by an independent analytical procedure (Table I). One advantage of this detection method is that the glycopeptide products can be used to determine the concentration and composition of the dolichol-linked oligosaccharides in the eluate fractions. Consequently, we need not metabolically label the dolichol-linked oligosaccharides prior to chromatography. Alternatively, the OS-PP-Dol compounds in the eluate fractions can be detected with a ConA binding assay, as described here, or by the inclusion of 3H mannose-labeled dolichol-linked oligosaccharides.
A single chromatography step using the aminopropyl silica HPLC column achieved a remarkable resolution of two dolichol-linked oligosaccharides that differ by one or more sugar residues. To our knowledge, previously described chromatographic methods for resolving individual OS-PP-Dol compounds are either less rapid and less versatile (Sharma et al., 1990), or are primarily applicable to the separation of small quantities of radiolabeled compounds (Wells et al., 1981
).
The procedure we have developed for the purification of dolichol-linked oligosaccharides from alg mutant yeast strains will yield pure bona fide intermediates in the assembly pathway. As shown here for the alg3 mutant, the steady-state dolichol-linked oligosaccharide pool isolated from an alg mutant yeast culture will likely contain several dolichol-linked oligosaccharide assembly intermediates. The use of substrate preparations that contain other assembly intermediates could complicate the biochemical analysis of a glycosyltransferase or the oligosaccharyltransferase. For example, if total dolichol-linked oligosaccharides isolated from the
alg3 mutant were used as an assay substrate for the mammalian OST, the initial rates of glycopeptide formation would primarily reflect transfer of Glc3Man7GlcNAc2 and Glc3Man5GlcNAc2 rather than Man5GlcNAc2.
As shown in Figure 6 a number of authentic assembly intermediates could be isolated from cultures of currently available alg mutants. The mannosyltransferase responsible for the addition of the terminal
-1,2 linked mannose residue to the
-1,6 dimannose antenna has not been identified. To date, only three yeast glycosyltransferase genes (ALG7, ALG1, and ALG2) have been identified that mediate assembly of Man5GlcNAc2-PP-Dol (Figure 6). The alg1 mutant accumulates GlcNAc2-PP-Dol, and the alg2 mutant accumulates a mixture of Man1GlcNAc2-PP-Dol and Man2GlcNAc2-PP-Dol (Huffaker and Robbins, 1982
, 1983; Jackson et al., 1989
, 1993). Large-scale isolations of pure dolichol-linked oligosaccharides from the alg1 and alg2 mutants should be feasible using organic solvent extraction procedures that are appropriate for the early assembly intermediates (Huffaker and Robbins, 1982
).
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We have confirmed and extended these observations by analyzing the steady-state pool of dolichol-linked oligosaccharides in a alg3 strain. We observed that Man5GlcNAc2-PP-Dol, Glc3Man5GlcNAc2-PP-Dol, and Glc3Man7GlcNAc2-PP-Dol were present in a 12:2.5:1 ratio. Glc3Man7GlcNAc2-PP-Dol was probably not detected in previous studies due to the low abundance of this compound and the more complex N-linked oligosaccharide profile that was obtained from cultures of the slightly leaky alg3 mutant (Verostek et al., 1991
, 1993). However, neither Glc3Man7GlcNAc2-PP-Dol nor Glc3Man5GlcNAc2-PP-Dol were detected in a more recent analysis of 3H mannose-labeled dolichol-linked oligosaccharides isolated from the
alg3 strain (Burda and Aebi, 1999
). One possible explanation for this discrepancy is that a brief pulse-label with 3H mannose may underestimate the quantity of the glucosylated donors synthesized by the
alg3 yeast strain.
Our results demonstrate that the Alg12p -1,6 mannosyltransferase can utilize Man5GlcNAc2-PP-Dol as a substrate, albeit with very low efficiency. Apparently, the Alg8p and Alg10p glucosyltransferases are much less sensitive than the Alg6p glucosyltransferase to the absence of the
-1,3-
-1,2 and
-1,6-
-1,2 dimannose antennas, as significant amounts of the monoglucosylated and diglucosylated intermediates containing 6, 7, or 9 hexose units were not detected. The absence of Man7GlcNAc2-PP-Dol from the
alg3 dolichol-linked oligosaccharides suggests that the presence of the
-1,6-
-1,2 antenna serves as the primary recognition determinant for the Alg6p glucosyltransferase. Thus, the lack of the
-1,3-
-1,2 dimannose antenna on Man5GlcNAc2-PP-Dol may reduce recognition by the Alg6p glucosyltransferase in an indirect manner by failing to serve as an adequate substrate for the Alg12p
-1,6 mannosyltransferase which initiates synthesis of the
-1,6-
-1,2 dimannose antenna.
The OST endpoint assay is a very sensitive method to determine dolichol-linked oligosaccharide composition due to the high specific activity of the iodinated peptide acceptor (15,00025,000 cpm/pmol). Recently, considerable attention has been focused on a group of diseases known as congenital disorders of glycosylation (CDG), formerly referred to as carbohydrate deficient glycoprotein syndrome (CDGS), which are caused by defects in the assembly or processing of N-linked oligosaccharides. For example, CDG-1d is caused by a defect in the Dol-P-Man-dependent Man5GlcNAc2-PP-Dol:-1,3 mannosyltransferase encoded by the human ALG3 gene (Korner et al., 1999
). Composition analysis of dolichol-linked oligosaccharides isolated from CDG cell lines should be feasible using the OST endpoint assay followed by analytical HPLC. Moreover, the biochemical characterization of the enzyme defects would be aided by the availability of purified dolichol-linked oligosaccharides.
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Materials and methods |
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Sixty grams of freshly excised pancreas was typically chilled to 0°C by immersion in ice-cold homogenization buffer (20 mM TrisCl [pH 7.5], 0.25 M sucrose, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1x protease inhibitor mixture [as defined in Kelleher and Gilmore, 1994] and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]). For the dolichol-linked oligosaccharide isolation shown in Figure 1C and 1D, the porcine pancreas was initially chilled in situ using ice-cold Krebs-Ringer buffer. The isolation of a crude microsome fraction from the porcine pancreas occurred in a cold room to minimize glycosidase activities. The chilled tissue was minced and then mixed with 1.3 ml of the homogenization buffer per g of tissue. The 136 ml of resuspended tissue was homogenized in several batches using 25 strokes of a Potter-Elvehjem homogenizer as described for the canine pancreas rough microsome preparation (Walter and Blobel, 1983
). The homogenate was centrifuged for 10 min at 10,000 x g in a Sorvall SS34 rotor to remove unbroken cells, nuclei, and large organelles. A floating fat layer was removed with a spatula and discarded before the supernatants were collected and pooled. The supernatant was diluted to 140 ml with homogenization buffer and divided into four 35-ml portions. Two portions were incubated at 37°C for 60 min in glass bottles to permit hydrolysis of terminal glucose residues on the OS-PP-Dol by endogenous RER glucosidases. All four portions were frozen in liquid nitrogen for storage at 80°C. The crude microsomes that were not exposed to the 37°C incubation served as starting material for the isolation of Glc3Man9GlcNAc2-PP-Dol, while the 37°C treated microsomes served as starting material for the isolation of Man9GlcNAc2-PP-Dol.
The four frozen membrane preparations were rapidly thawed in a 37°C water bath, adjusted to 35 ml with cold homogenization buffer if necessary, and transferred into four 150-ml glass round-bottom centrifuge tubes. The membrane preparations were mixed with 50 ml of cold (4°C) CHCl3:CH3OH (3:2) and homogenized with six 10-s bursts of a Brinkman Polytron homogenizer at maximum speed. After homogenization, an additional 65 ml of cold (4°C) CHCl3:CH3OH (3:2) was added to each centrifuge tube and the contents mixed by vigorous shaking. After centrifugation for 15 min at 4°C and 2500 rpm in a Sorvall HS4 swinging bucket rotor, the upper aqueous and lower organic phases were removed by aspiration and discarded. The particulate interface wafers (0.4 cm thick) were resuspended in 75 ml of room-temperature CHCl3:CH3OH (3:2) containing 1 mM MgCl2 by polytron homogenization as described above and adjusted to 150 ml with the same solvent mixture. Following centrifugation for 15 min at 4°C and 2500 rpm in a Sorvall HS4 swinging bucket rotor, the supernatant was removed and the precipitate was resuspended in 75 ml of CH3OH:4 mM MgCl2 (1:1) by polytron homogenization, diluted to 150 ml with CH3OH:4 mM MgCl2 (1:1) and centrifuged as above at 4°C. The preceding wash with aqueous methanol was repeated a second time. The precipitate was resuspended by polytron homogenization in 75 ml of CHCl3:CH3OH:H2O (10:10:3), diluted to 150 ml with the same solvent mixture, and incubated for 15 min at 22°C prior to centrifugation for 15 min at 22°C and 2500 rpm in a Sorvall HS4 swinging bucket rotor. The supernatant fraction containing the OS-PP-Dol was removed and saved. Each of the precipitates was reextracted at 37°C with 100 ml of CHCl3:CH3OH:H2O (10:10:3). The supernatants from the second extraction were combined with the first extracts, and the solvents were evaporated under a stream of nitrogen in a fume hood. The dried residues were dissolved in CHCl3:CH3OH:H2O (10:10:3) and pooled to obtain one 7.5-ml sample for each of the two crude membrane preparations (± 37°C incubation). The resuspended extracts were transferred to conical glass centrifuge tubes for a 15-min spin at 1000 x g at 22°C to remove any insoluble material. The clarified extracts were transferred to new glass tubes with Teflon caps, adjusted to 7.5 ml CHCl3:CH3OH:H2O (10:10:3) and stored at 20°C. As determined with an OST endpoint assay, each preparation was about 3.7 µM OS-PP-Dol, which would correspond to a yield of 0.93 nMol of OS-PP-Dol per g of tissue.
Dolichol-linked oligosaccharide isolation from yeast
Lipid-linked oligosaccharides were isolated from a crude microsomal membrane fraction (10,000 x g supernatant fraction). Briefly, the alg3 mutant (YG248) (Aebi et al., 1996
) was grown at 25°C to a density of 2.2 (A600) in 10.5 L of minimal media (-His) supplemented with adenine (40 µg/ml) using a Bioflo 2000 fermenter. The yeast cells were collected by centrifugation, washed once, and disrupted as described previously (Kelleher and Gilmore, 1994
) with 210 ml of glass beads in 190 ml of buffer (50 mM TEA-OAc [pH 7.5], 0.25 M sucrose, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1x protease inhibitor mixture, and 0.1 mM PMSF). After the glass beads were removed by passage through a nylon mesh filter, a 10,000 x g supernatant fraction was prepared as described (Kelleher and Gilmore, 1994
) and adjusted to a total volume of 210 ml. Dolichol-linked oligosaccharides were isolated from the 10,000 x g supernatant fraction as described above for the porcine pancreas, with the exception that all six 35-ml aliquots were used for the isolation without the 37°C preincubation.
HPLC of dolichol-linked oligosaccharides
Individual dolichol-linked oligosaccharides in the extracts prepared from vertebrate tissue or yeast cultures were resolved by chromatography on a 24.3-ml aminopropyl silica HPLC column purchased from Varian/Rainin. The column consisted of a 1 x 6 cm guard column (R0080710C5) followed by a 1 x 25 cm preparative column (R00807010G5), both of which were packed with Microsorb aminopropyl silica (5 µ particle, 100 Å pore) in Dynamax hardware. Solvent resistant valves and fittings were used for all connections. Solvent A was CHCl3:CH3OH:H2O (10:10:3), and solvent B was CHCl3:CH3OH:2 M NH4OAc (pH 7.2) (10:10:3). Prior to chromatography, the column was first washed at a flow rate of 4 ml/min with 250 ml of solvent B followed by 250 ml of solvent A. The flow rate of solvent A was reduced to 2 ml/min for an additional 10 min of equilibration. The 4-ml sample applied to the column contained as much as 15 nmol of a crude OS-PP-Dol preparation dissolved in solvent A. The sample was applied at a flow rate of 2 ml/min, and the column washed with solvent A for 10 min at this flow rate before increasing the flow rate to 4 ml/min over a 1-min period. Flow-through and wash fractions of 40 ml each were collected during a 60-min period after sample injection. Less than 0.2% of the dolichol-linked oligosaccharide was recovered in the flow-through and wash fractions. At the 60-min time point, a 320-ml linear gradient of 040% solvent B was initiated at a flow rate of 4 ml/min. This gradient corresponds to an increase of 1.3 mM NH4OAc per 4-ml fraction of eluate. At the 140-min time point, a 1-min gradient between 40% and 100% B was followed by a 15-min wash with 100% B.
Oligosaccharyltransferase endpoint assay
The yeast OST was purified by a modification of a previously described procedure (Kelleher and Gilmore, 1994), using a strain expressing a hexahistidine-tagged OST1 subunit. The strain construction and purification procedure will be described elsewhere (Karaoglu, Kelleher, and Gilmore; manuscript in preparation). The iodinated tripeptide acceptor N
-Ac-Asn-[125I]Tyr-Thr-NH2 (15,00025,000 cpm/pmol) was prepared as described previously (Kelleher et al., 1992
). The 50 µl endpoint assays to estimate OS-PP-Dol concentration and to yield products for composition analysis contained 1015 fmol of purified yeast OST, 5 µM N
-Ac-Asn-[125I]Tyr-Thr-NH2, approximately 2.518 pmol of OS-PP-Dol, 50 mM Tris-Cl (pH 7.4), 2.5 mM NaCl, 2 mM MnCl2, 3 mM MgCl2, 0.006% digitonin, 1.35 mM phosphatidylcholine, 1 mM DTT, 15% DMSO, 5% glycerol, and 0.13 mM EDTA. For more precise composition analysis, the OST endpoint assays were supplemented with 1.3 mM each of deoxynojirimycin and deoxymannojirimycin. The OST endpoint assays were terminated after 4872 h by the addition of 0.1 ml of ice-cold 9.4% Nikkol (octaethyleneglycol-mono-N-dodecyl ether; Nikko Chemicals, Inc.), 11.3 mM EDTA. The assay was diluted with 1 ml of ice-cold 50 mM TrisCl (pH 6.7), 1 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, 0.3% Nikkol, and the glycopeptide products collected during a 20-min end-over-end incubation with 100 µl of a 1:1 suspension of ConA Sepharose beads equilibrated in ConA-HS buffer (50 mM TrisCl [pH 6.7], 1 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, 0.02% Nikkol). The beads were collected by centrifugation and washed four times with 1 ml of Con A-HS buffer. Glycopeptide products were quantified by gamma counting and corrected for nonspecific binding of N
-Ac-Asn-[125I]Tyr-Thr-NH2 to the ConA beads in control assays that lacked either enzyme or OS-PP-Dol.
HPLC analysis of glycopeptides
For oligosaccharide composition analysis, the ConA Sepharose beads were washed twice with 1.4 ml of cold water, after which the glycopeptides were eluted by two rapid extractions at 25°C with 0.75 ml of 60% acetonitrile, 0.2 N HCl. The combined eluates were dried in a Speed-Vac and resuspended in 300 µl of 70% acetonitrile, 3% acetic acid adjusted to pH 5.5 with triethylamine (buffer A). The HPLC conditions used for resolving neutral glycopeptides on the basis of oligosaccharide size are based on the method of Mellis and Baenziger (1983). The HPLC column was a Varian/Rainin aminopropyl silica column composed of a 0.46 x 6 cm guard column (R0080700G5) followed by a 0.46 x 25 cm analytical column (R0080725C5), both of which were packed with Microsorb aminopropyl silica (5 µm particle, 100 Å pore). Buffer B for the HPLC column was 3% acetic acid in H2O adjusted to pH 5.5 with triethylamine. The concentration of triethylamine in buffers A and B is about 90 mM and 432 mM, respectively. Samples were injected into the column at a flow rate of 1 ml/min, followed by a 10-min wash with buffer A. The glycopeptides were eluted with a 60-min linear gradient between 0% and 40% buffer B at a flow rate of 1 ml/min. One hundred 0.5-ml fractions were collected and the quantity of radiolabeled glycopeptide determined by gamma counting. Between chromatography runs, the column was washed with 15 ml of buffer B.
Solid state ConA binding assay
Unlabeled OS-PP-Dol in HPLC fractions was detected using a semiquantitative ConA binding assay. Known quantities of OS-PP-Dol (17 pmol) in CHCl3:CH3OH:H2O (10:10:3) or 100-µl aliquots of eluate fractions from the aminopropyl silica column were dried in 12 x 75 mm disposable round-bottom glass tubes in a Savant Speed-Vac. The samples were incubated for 20 min at room temperature in 0.5 ml of ConA-MS buffer (20 mM TrisCl [pH 6.7], 500 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2) adjusted to 2% bovine serum albumin as a blocking agent. The blocking solution was removed by aspiration and replaced with 0.5 ml of ConA-MS buffer that contained 0.5 µg of ConA coupled to horseradish peroxidase (Sigma L-6397). After 30 min of incubation at 25°C, the ConA solution was removed by aspiration and the tubes were washed three times with 1 ml of ConA-MS buffer. The assay tubes were placed in a foam rubber test tube rack with the tubes protruding slightly. After adding 1 ml of ConA-MS buffer, the test tube rack was transported to a dark room, the rack was inverted, and the tubes allowed to drain for 1 min. Aliquots (250 µl) of freshly prepared ECL reagent (KPL 50-62-00) were then added by repipet to a maximum of 50 tubes at a time. After 1 min, the tubes were placed on an 8'' x 10'' sheet of Kodak XAR film for 10120-s exposures. A 30-s exposure allowed detection of 1 pmol of OS-PP-Dol per tube. The assay relies on the binding of the OS-PP-Dol to the glass and the insolubility of OS-PP-Dol in aqueous solutions lacking detergent and is not affected by the presence of 200 mM NH4OAc in the assay sample.
TLC of dolichol-linked oligosaccharide
Water-resistant 20 x 20 cm silica thin-layer plates with a 0.2 mm coating on an aluminum support (EM-16487-1) were purchased from VWR. The TLC plates were prerun in a TLC tank that had been preequilibrated with CHCl3:CH3OH:H2O (10:10:3) and dried. Samples from the preparative HPLC columns containing 50150 pmol of OS-PP-Dol in CHCl3:CH3OH:00.1 M NH4OAc (10:10:3) were dried in a Savant Speed-Vac, redisolved in 15 µl of CHCl3: CH3OH:H2O (10:10:3) and spotted 2.5 cm from the bottom of a TLC plate. The TLC plate was developed in CHCl3:CH3OH:H2O (10:10:3) until the solvent front had risen 1112 cm from the origin (
6080 min).
Glucose and mannose containing compounds on the TLC plate were detected with a fluorescein isothiocyanate (FITC) derivative of concanavalin A (Sigma, C-7642). The TLC plates were dried and placed in a polypropylene dish containing 50 ml of ConA-MS buffer that contained 500 µg of ConA-FITC. After 80 min of incubation at 25°C, excess ConA was removed by five successive 10-min washes of the TLC plate with 150 ml of ConA-MS buffer. After excess moisture was removed from the TLC plate by placing it on filter paper, the plate was exposed to broad band UV illumination (290365 nm) within a Fluoro-S Multi-Imager. The emitted light was collected for 30180 s after passage through a 530DF60 band pass filter (495555 nm). The images were exported from the BioRad MultiAnalyst software to Adobe Photoshop. When the ConA detection method for TLC plates worked well, the TLC could be incubated for 48 h in ConA buffer-MS without signal loss. The unexpectedly low (75 pmol of OS-PP-Dol) and somewhat variable sensitivity of this detection method suggests that the TLC plate may interfere with ConA detection of OS-PP-Dol or that the OS-PP-Dol may either hydrolyze during chromatography or incubation in the ConA-MS buffer.
The TLC plates not exposed to ConA-FITC were stained with anisaldehyde to visualize as many compounds as possible in the crude OS-PP-Dol preparation. Preliminary experiments showed that no additional compounds were detected by UV absorption of F254-impregnated silica gel plates or by an iodine vapor staining, alone or in combination with an anisaldehyde stain. The dried TLCs were sprayed until saturated with a freshly prepared solution containing 1 ml of 4-methoxybenzaldehyde (Sigma A-0519), 100 ml of glacial acetic acid, and 2 ml of sulfuric acid (Lewis and Smith, 1972). The TLC was baked in an oven at 105°C for 4560 min to allow color development. The images were captured using Microtek Scanmaker III and imported into Adobe Photoshop where they were converted into grayscale images for publication. 50 pmol of OS-PP-Dol could be detected by anisaldehyde staining of TLC plates.
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