3 Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, NY 12201, USA and 4 Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201, USA
Received on April 12, 2002; revised on June 20, 2002; accepted on July 5, 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: glycan 1H NMR/glycoprotein processing/N-linked oligosaccharides/S. cerevisiae/yeast alg mutants
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutants with lesions late in the yeast OSL pathway display no selectable phenotype. However, recently, Aebi and co-workers isolated the late-acting genes ALG3, ALG6, ALG8, ALG9, and ALG10 by rescuing through complementation their respective synthetically lethal phenotypes occurring in conjunction with mutations in subunits of OST (Stagljar et al., 1994; Aebi et al., 1996
; Burda et al., 1996
; Burda and Aebi, 1998
; Reiss et al., 1996
). This screening technique, in addition to 3H-Man suicide (Huffaker and Robbins, 1982
, 1983) and sodium vanadate resistance (Dean, 1995
), have identified a mutant in nearly every step in Glc3Man9GlcNAc2-PP-Dol synthesis (Scheme S1A) and its subsequent processing to "mannan." The remaining unidentified N-glycan pathway genes, not found by the more conventional methodologies, are now being isolated by applying homology-searching algorithms to the annotated S. cerevisiae genome (Cherry et al., 1997
).
|
Recently, we showed in alg9 yeast that the central-arm
1,3-linked Man residue 7 added by Alg3p (Scheme S1) potentiates the Alg6p, Alg8p, and Alg10p glycosyltransferases, the Gls1p/Cwh41p and Gls2p glucosidases I and II, and the Golgi Ochlp initiation of
1,6-Man outer chain (Cipollo and Trimble, 2000
). Here, we extend our understanding of the role individual Man residues in OSL play in subsequent N-glycan processing by defining the structure of alg12
N-glycans and the fate of
1,2-linked Man residue 10 added by Alg9p (Scheme S1A). Interestingly, invertase and protease A (PrA) are hypoglycosylated in alg12
, leading to their instability, which provides a practical indication of the role proper synthesis and processing of N-glycans plays in glycoprotein function (Cipollo and Trimble, 2002
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of alg12 invertase
By monitoring PrA activity after derepression for invertase and adding pepstatin A to 3 µM when protease activity began to rise (Cipollo and Trimble, 2002), alg12
yeast transformed with the pRB58 plasmid (Verostek et al., 1991
) overproduced SUC2 invertase at a level exceeding 700 IU/g (wet weight). This yielded a high specific activity external invertase (68 mg, 3670 U/mg protein) with an overall recovery of 60% from 635 g of cells.
A western blot of wild-type, alg12-, and alg9
-secreted invertases revealed wild-type external invertase to migrate as a diffuse band with an average apparent mass of ~120 kDa, whereas external invertases from alg12
and alg9
were progressively more heterogeneous in mass with average weights of 110 kDa and 95 kDa, respectively (data not shown). Bio-Gel P-4 size-exclusion chromatography of the peptide-N-glycosidase F (PNGase F)released glycans from the alg12
invertase preparation provided four major pools, labeled A through D in Figure 1, which eluted on the calibrated column in volumes consistent with Hex7GlcNAc2 (fractions 121125), Hex8GlcNAc2 (fractions 114120), Hex9.5GlcNAc2 (fractions 111113), and Hex11GlcNAc2 (fractions 106110), respectively.
|
|
|
|
|
|
|
Pool B: Hex89GlcNAc2. The anomeric proton intensity in pool B was 8.27 mol (Table I), indicating that 73% of the isomers present were Hex8GlcNAc2, and 27% were Hex9GlcNAc2, in good agreement with the MALDI-TOF MS distribution estimated in Figure 4B to be 80% Hex8GlcNAc2 and 20% Hex9GlcNAc2. C1-H proton intensity in excess of the core Man6 reference isomer (Scheme S1B and Table I) was present at chemical shifts at 4.89 ppm (0.09 mol), 5.04 ppm (1.18 mol), 5.135.15 ppm (0.91 mol), and 5.25 ppm (0.10 mol). Core residue 7 was found distributed as 0.85 mol at 5.41 ppm, and the remaining 0.15 mol at 5.11 ppm (Table I). Thus, as assigned and as documented by the 2D DQF COSY cross-peak at 5.41 (C1-H)/4.08 (C2-H) ppm (Figure 4B), 85% of the isomers present have 1,2-Man 10 on Man 7.
At 4.14 ppm the total proton intensity was 1.42 mol (Table I), which included 1.00 mol from residue 4 and 0.42 mol from residue 3 due to the through-space effect on 3 caused by 5s 6-O-substitution with residue 12. Thus, 42% of pool-B isomers contain residue 12. The 0.09 mol of resonance intensity seen at 4.92 ppm (Table I) is from unsubstituted 12. The assignment was confirmed by a low cut in the C1-H/C2-H region of the DQF COSY spectrum, which revealed a cross-peak centered at 4.92 (C1-H)/3.91(C2-H) ppm (not seen in Figure 4B; Trimble and Atkinson, 1992; Cipollo and Trimble, 2000
). Subtracting the 0.09 mol of unsubstituted 12 from the 0.42 mol total for 12 leaves 0.33 mol of 12 2-O-substituted by 13. These allocations necessitate that in addition to the core residues 15, 7, 8, and 11, the following can be made as partial structural assignments: 9% have terminally linked 12, 33% have 12 and its 2-O-substituent residue 13, and 58% are minimally processed core type with no added 12 or 13.
At 5.04 ppm the 1.18 mol of proton intensity is divided between terminal and 3-O-substituted 1,2-Man, whose presences are verified by the 2D DQF COSY cross-peaks seen at 5.04 (C1-H)/4.06(C2-H) ppm and 5.03(C1-H)/4.22(C2-H) ppm, respectively (Figure 4B; Haeuw et al., 1991
; Verostek et al., 1993b
; Cipollo and Trimble, 2000
). Of this resonance intensity, 0.85 mol was assigned to residue 10, leaving 0.33 mol of this signature to assign. For the Hex89GlcNAc2-sized compounds studied here, added proton resonance intensity, above that seen for residue 10, can only come from residue 13 (Scheme S1D), which 2-O-substitutes residue 12 (Trimble et al., 1991
; Trimble and Atkinson, 1992
; Cipollo and Trimble, 2000
). This indicates that 33% of pool isomers have residue 13, coinciding with the amount assigned using the C2-H intensities at 4.14 ppm.
Between 5.10 and 5.14 ppm, 1.06 mol of resonance intensity in excess of that attributed to the core Man6GlcNAc2 was integrated. Of that 1.06 mol, 0.15 mol of intensity was present at 5.11 ppm. Subtracting the 0.15 mol of unsubstituted 7s intensity at 5.11 ppm (see previous discussion) from the 1.06 mol of signal in the 5.105.15 ppm region leaves 0.91 mol to assign. A 2D DQF COSY cross-peak seen at 5.14 (C1-H)/4.03(C2-H) ppm (Figure 4B) is characteristic of 1,6-Man 12 when 2-O-substituted by residue 13 (Verostek et al., 1993b
; Cipollo and Trimble, 2000
) and accounts for 0.33 mol of the remaining 0.91 mol of intensity, leaving 0.58 mol to assign. The final 2D DQF COSY cross-peak detected from anomeric protons between 5.10 and 5.15 ppm was at 5.14(C1-H)/4.06(C2-H) ppm (Figure 4B), due to terminally linked
1,3-Man, and accounts for the remaining 0.58 mol of proton intensity. In the compounds with the Man7GlcNAc2 core studied here, this resonance can arise from mannoses 14, 15, 16, and 18 (Scheme S1D; Cipollo and Trimble, 2000
).
At 5.25 ppm 0.10 mol of resonance intensity was integrated. The 2D DQF COSY spectrum reveals a cross-peak at 5.25- (C1-H)/3.56(C2-H) ppm, confirming the presence of residue G1 (Trimble and Atkinson, 1986; Verostek et al., 1993b
; Cipollo and Trimble, 2000
). This indicates that 10% of the pools isomers retained the innermost
1,3-linked Glc residue on the lower-arm Man residue 11 (Scheme S1A).
At 4.22 ppm 1.26 mol of C2-H intensity was integrated. Of this, 0.58 mol is from 3 in the absence of 12 as assigned. Another 0.10 mol is from residue G1, leaving 0.58 mol attributed to the fractional 3-O-substitution by residues 14, 15, and/or 16 of residues 10, 11, and/or 13 (see Scheme S1D), which is in agreement with the assignment of 0.58 mol of 1,3-Man residues at 5.14 ppm derived earlier. Of the 0.58 mol of Man6GlcNAc2 core in pool B that does not have residue 12 (Scheme S1B), 0.48 mol of 10 and an equal amount of the terminal
1,3-Man residue 14/15, are assigned, defining 48% of the pool as isomer 8a (Scheme S2). The remaining 0.10 mol of 3-O-substituted Man, 0.10 mol of 10, and 0.10 mol of G1 are assigned to isomer 8b as 10% of the pool B isomers. This accounts for all of the 3-O-substituted Man C2-H at 4.22 ppm and leaves 0.27 mol of the 0.85 mol of 10 to assign. For the 33% of the pool components with residues 13 and 12 as assigned, 15% are without residue 10 defining glycan 8c. The remaining 18% of the isomers with residues 12 and 13 have residue 10, assigning isomer 9a (Scheme S2), which leaves 0.09 mol of 10 to assign. Isomer 8d with terminally linked Man 12 represents 9% of pool B and consumes 0.09 mol of remaining residue 10 (Scheme S2).
The HPAEC profile of the pool B gave a distribution of 49%, 22%, 22%, and 7% of total area with a pronounced shoulder on the peak eluting at 16 min (Figure 3B). This is consistent with the presence of two isomers having similar elution characteristics. Thus the 1H NMR assignments are in close agreement with the isomer distribution predicted by HPAEC analysis.
Pool C: Hex910GlcNAc2. The total Man and Glc anomeric proton intensity of pool C was 9.35 mol, which corresponds to 65% Hex9GlcNAc2 and 35% Hex10GlcNAc2 (Table I). These values are in close agreement with the MS profile, which estimated 60% Hex9GlcNAc2 and 40% Hex10GlcNAc2 (Figure 2C). In pool C the proton intensity of the Man6 core (Scheme S1B) was 3.35 mol, which included 0.17 mol between 4.89 and 4.92 ppm for unsubstituted 1,6-Man residue 12 (see Scheme S1D); 1.35 mol at 5.04 ppm for 2-O-linked Man residues 10 and 13; 1.79 mol at 5.14 ppm for
1,3-Man residues 14, 15, 16 and/or 18; and
1,6-Man 12 when 2-O-substituted with residue 13. The C2-H proton intensity at 4.22 ppm was 1.58 mol, and the intensity found at 4.14 ppm equaled 1.71 mol (Scheme S1B and Table I).
The anomeric proton intensity at 5.41 ppm for 2-O-substituted 7 was 0.81 mol, which was verified by the strong 2D DQF COSY cross-peak at 5.41(C1-H)/4.10(C2-H) ppm (Figure 4C), indicating that 80% of pool C isomers have residue 10. The remaining 0.19 mol of residue 7s anomeric proton was present at 5.11 ppm, characteristic of its unsubstituted form.
At 4.14 ppm 1.71 mol of C2-H proton intensity was found. As described for previously assigned isomers, this signal intensity is from residues 3 (when 12 is present) and 4. Because 4 contributes 1.00 mol of intensity, 3 contributes the remaining 0.71 mol, indicating that 71% of the pool isomers contain residue 12. The 0.17 mol of added signal seen at 4.92 ppm means that 17% of the pool isomers had an unsubstituted residue 12, verified by a low cut of the 2D DQF COSY spectrum, which revealed the 4.92 (C1-H)/3.98(C2-H) ppm cross-peak (seen in Figure 4C; Verostek et al., 1993b; Cipollo and Trimble, 2000
). By difference (0.710.17 mol), 0.54 mol of 12 is 2-O-substituted with 13, whose cross-peak is seen at 5.14 (C1-H)/4.02(C2-H) ppm in Figure 4C. This allows the following partial structural assignments: 54% of the pool glycans contain 12 and 13, 17% contain terminally linked 12, and 29% are devoid of 12 and 13 (Table I and Scheme S1).
The 1.35 mol anomeric proton intensity seen at 5.04 ppm (Table I) is divided between terminal and 3-O-substituted 1,2-Man residues 10 and 13. The 2D DQF COSY J1,2 cross-peaks at 5.04 (C1-H)/4.07(C2-H) ppm and 5.03(C1-H)/4.22(C2-H) ppm (Figure 4C) verify the presence of both unsubstituted and 3-O-substituted
1,2-Man residues, respectively (Trimble and Atkinson, 1992
; Verostek et al., 1993a
,b; Cipollo and Trimble, 2000
). Because 0.81 mol is present as
1,2-Man 10, which is equal to the intensity of 2-O-substituted 7 at 5.41 ppm in Table I, by subtraction 0.54 mol of proton intensity is from terminal
1,2-Man 13. These assignments confirm the presence of 13 and 12 on 54% of the pool C isomers as calculated in the previous paragraph.
As observed from pool Cs 2D DQF COSY spectrum (Figures 4C and 5C), 1.79 mol of proton intensity above the Man6 core was present at 5.14 ppm and was distributed between two residue linkage types. The defining cross-peaks were found at 5.14 (C1-H)/4.02 (C2-H) ppm and 4.02 (C2-H)/3.92 (C3-H) ppm for the 2-O-substituted 1,6-linked residue 12 (Trimble and Atkinson, 1992
) and 5.14 (C1-H)/4.07 (C2-H) ppm and 4.07 (C2-H)/3.88 (C3-H) ppm for terminal
1,3-Man. Subtracting 0.54 mol of 13 from the 1.79 mol of intensity at 5.14 ppm defines 1.25 mol of
1,3-Man.
At 5.25 ppm, 0.04 mol of proton intensity was detected for G1 (Table I). Although not apparent in Figure 4C, this assignment is confirmed by a near baseline cut in pool Cs 2D DQF COSY spectrum, which revealed the defining cross-peak at 5.25 (C1-H)/3.54 (C2-H) ppm (Trimble and Atkinson, 1986; Verostek et al., 1993b
).
Of the 1.58 mol of C2-H intensity seen at 4.22 ppm, 0.29 mol was from residue 3 in structures lacking residue 12, leaving 1.25 mol arising from 3-O-substituted residues 14, 15, and 16, and 0.04 mol from residue 11 3-O-substituted by G1. To a portion of pool C isomers with residues 12 and 13 (54%), we assign 0.35 mol of 3-O-substituted Man and an equal amount of 5.04 ppm resonance from the 0.81 mol of residue 10, defining 35% of the pool as the major isomer 10a. This leaves 0.90 mol of 3-O-substituted Man and 0.46 mol of Man 10 to assign. To the remaining 19% of pool Cs isomers having residues 12 and 13 was assigned 0.19 mol of terminal 1,3-Man, defining pool B isomer 9b, and leaving 0.71 mol of resonance at 4.22 ppm, equal to the remaining
1,3-Man to assign. Note that isomer 9b accounts for all of
1,3-Man 7 as a terminal residue, whose resonance was detected at 5.11 ppm as defined earlier. To 17% of the pool isomer with unsubstituted residue 12 (the 0.17 mol signal at 4.92 ppm in Table I) was assigned 0.17 mol of both
1,3-Man and residue 10 to give isomer 9c. This leaves 0.54 and 0.29 mol, respectively, of each residue to assign. To the 29% Man6 core structure (Scheme S1B) that did not have residues 12 or 13 described earlier, we assigned 0.25 mol of the remaining 10 and 0.50 mol of
1,3-Man, defining isomer 9d. This leaves 0.04 mol each of G1,
1,3-Man, residue 10, and the Man6 glycan, which together define isomer 9e (4%), thus completing the assignment of the pools components. All structures can be seen in Scheme S2. The number and distribution of isomers in the pool as derived by proton intensity allocation (Table I) are in good agreement with the isomer number and distribution predicted by HPAEC and estimated Hex9/Hex10 MS intensities (60/40).
Pool D: Hex1011GlcNAc2. pool. The Man and Glc anomeric proton intensity above the Man6 core in pool D (Scheme S1 and Table I) was 4.60 mol, giving a total of 10.60 mol of proton intensity. This indicates that the pool contains 60% Hex11GlcNAc2 and 40% Hex10GlcNAc2, which is in close agreement with the MALDI-TOF MS prediction of 70% Hex11GlcNAc2 and 30% Hex10GlcNAc2 (Figure 2D). The increased C1-H resonance intensity above that provided by the core Man6GlcNAc2 was observed at 5.25 ppm (trace), 5.14 ppm (2.70 mol), 5.04 ppm (1.75 mol), and 4.92 ppm (0.15 mol).
The 0.90 mol of signal at 5.41 ppm is from core residue 7 2-O-substituted by 10, which is verified by the characteristic 2D DQF COSY cross-peak at 5.41 (C1-H)/4.10 (C2-H) ppm (Figure 4D). Subtracting 0.90 mol of residue 10 from the 1.75 mol of signal at 5.04 ppm for 1,2-linked Man leaves 0.85 mol, which is assigned to
1,2-Man 13 substituting 12. This assignment is supported by the strong 2D DQF COSY cross-peak at 5.04 (C1-H)/4.07 (C2-H) ppm (Figure 4D), the signature of terminal
1,2-Man. Another cross-peak at 5.03 (C1-H)/4.22 (C2-H) ppm was observed for 3-O-substituted
1,2-Man (Figure 4D), indicating that a portion of the pools isomers have
1,3-Man residues 14, 15, and/or 16. Thus, 90% of pool Ds glycans have residue 10 and 85% have
1,2-Man 13, which 2-O-substitutes Man 12.
At 4.91 ppm, 0.15 mol of resonance intensity in excess of the Man6 core residue 4 was integrated. The assignment is supported by the presence of a 2D DQF COSY cross-peak of low intensity at 4.91 (C1-H)/4.02 (C2-H) ppm (not apparent in Figure 4D). Thus all of pool Ds isomers have residue 12; 15% unsubstituted and 85% 2-O-substituted with 13 (Table I).
The 2.80 mol of resonance at 5.115.15 ppm that provided by the core Man6 structure includes 0.10 mol from core residue 7 not substituted by 10. This amount is derived by subtracting 0.90 mol of 2-O-substituted 7 at 5.41 ppm from unity, in agreement with integration of ~0.10 mol of resonance intensity at 5.11 ppm in the 1D NMR spectrum (Table I). An additional 0.85 mol of intensity in this chemical shift region of the spectrum is from 2-O-substituted 12 (described earlier). The remaining intensity at 5.115.15 ppm (1.85 mol) is assigned to 1,3-linked residues 14, 15, 16, and 18 (Scheme S1D). A strong cross-peak in the 2D DQF COSY spectrum of pool D at 5.14 (C1-H)/4.06 (C2-H) ppm (Figure 4D) confirms the presence of these terminal
1,3-linked residues. At 4.22 (C2-H)/4.01 (C3-H) ppm (Figure 5D), a low-intensity J2,3 cross-peak is present, indicating that a small amount of 14, 15, and/or 16 is 3-O-substituted with residue 18 (Verostek et al., 1993b
; Cipollo and Trimble, 2000
).
Note in the J2,3 region of the 2D DQF COSY spectrum the absence of a cross-peak for residue 3 at 4.22 (C2-H)/4.01 (C3-H) ppm (compare Figures 5AD), thus confirming that all of pool Ds isomers have residue 12. With 3s C2-H shifted upfield, all of the assigned 4.22 ppm signal (1.85 mol) must be from 3-O-substituted 1,2-Man and
1,3-Man residues. The 2.00 mol C2-H resonance intensity at 4.14 ppm is for the C2-Hs of 3 and 4, as expected (Table I). Distribution of the integrated protons and a Hex1011GlcNAc2 size constraint allow assignment of the two major isomers in pool D as 11a (60%) and 10a (15%) (Scheme S2). These structures account for 1.35 mol of resonance intensity at 4.22 ppm, leaving 0.50 mol to assign. Isomers 10b (15%) and 10c (10%) account for the remaining 0.5 mol of 4.22 ppm resonance intensity (see Scheme S2 for isomers). In addition, a trace amount of G1s anomeric proton was detected at 5.25 ppm in pool Ds 1D NMR spectrum (Table I), which leads to the assignment of a trace of isomer 11b. These assignments are in good agreement with the pools HPAEC profile, which gave a predicted isomer distribution of 60%, 16%, 10%, 10%, and 4% (Figure 3).
In vivo glucosylation of alg12 OSL
NMR-derived structures of secreted invertase glycans in alg12 yeast indicate that ~4% of Hex711GlcNAc2 isomers retained residue G1 (Scheme S2). To ascertain whether G1 was a product of compromised OSL glucosylation and impaired Glc trimming on nascent glycoproteins, as seen in alg3-1 yeast (Verostek et al., 1991
), or a remnant of full glucosylation and processing as seen in alg9
cells (Cipollo and Trimble, 2000
), alg12
cells were pulse-labeled for 2 min with [2-3H]Man in the absence (Figures 6AC) or presence (Figures 6DF) of the glucosidase inhibitor castanospermine (CST) and the label chased with unlabeled Man for 0, 1, or 10 min. The labeled glycans were released from glycoprotein pellets by endoglycosidase H (endo H) and analyzed by HPAEC as described in Materials and methods. The additional trace in Figure 6A and D (dashed line) is overlayed from a separate run of a [3H]Man7GlcNAc standard isolated from alg12
OSL and treated with endo H to remove one GlcNAc.
|
Comparison of the glycan profiles in Figures 6A and 6D implies that in alg12 essentially all of the OSL is fully glucosylated, and, following transfer to protein, the glucoses are rapidly and efficiently removed. This occurs so quickly, in fact, that during the 2-min pulse in the absence of CST (Figure 6A), one to three Glc residues were removed from nearly all the labeled glycans and a portion coelute with the [3H]Man7GlcNAc external standard (
). In the presence of CST, some of the Glc3Man7GlcNAc has already been elongated to larger species eluting by HPAEC at 3345 min (Figure 6D).
After only 1 min of chase with unlabeled Man almost all of glucosylated glycans were either deglucosylated (Figure 6B) or elongated to larger forms (Figure 6E). Since the t1/2 of protein secretion in yeast is about 5 min (Franzusoff, 1992), and no labeled ER forms of glycoproteins are seen after a 10-min chase (Franzusoff and Schekman, 1989
), the glycans found after the 10-min chase in Figures 6C and F are from glycoproteins trafficked to the Golgi and beyond. These constitute elongated species (the Vo glycans in Figure 1), which elute on column regeneration (Cipollo and Trimble, 2000
) and shorter glycans eluting from 7 to 40± min, which include the species present in Bio-Gel P-4 pools AD (Figures 1 and 3).
After 2 min of labeling in both the presence and absence of CST, a peak was seen in the HPAEC profile at 10 min (Figure 6A and D). Because no Glc trimming intermediates were seen between 10 and 26 min in the elution profile immediately after the 2-min pulse in the presence of CST (Figure 6D), it is likely this peak represents a small amount of Glc1Man7GlcNAc2 that was transferred directly to protein in the alg12 background under conditions in which Glc3Man7GlcNAc2-PP-Dol availability may have been limiting. Bio-Gel P-4 analysis of [3H]Man-labeled glycans from the OSL fraction from cells labeled during the pulse-chase experiment revealed that the relative amount of Glc3Man7GlcNAc2-PP-Dol was low (Figure 6A), whereas the amount of glycan eluting at 10 min was significant, consistent with Glc1Man7GlcNAc2-PP-Dol (Figure 6AC). However, the amount of labeled glycan eluting at 10 min in the HPAEC profiles was insufficient to structurally confirm this peak as Glc1Man7GlcNAc. Nevertheless, it is worth noting that Glc1Man7GlcNAc2 and Glc1Man8GlcNAc2 were among invertase glycans present in pools B and C (Scheme S2) at levels that could easily account for the [2-3H]Man-labeled glycan peak(s) eluting at 811 min in Figure 6.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Overexpression of Alg12p in the alg9 background forms, in addition to the alg9
Man6 OSL (Scheme S1B), a novel Man7 OSL, Man
1,2Man
1,2Man
1,3 (Man1,6(Man
1,3)Man
1,6)Man
1,4GlcNAc
1,4GlcNAc-PP-Dol, consistent with the ALG12 locus encoding the Man-P-Dol:Man7GlcNAc2-PP-Dol
1,6-mannosyltransferase that adds the upper-arm
1,6-linked Man residue 6 (Scheme S1A) to OSL (Burda et al., 1999
). Apparently, overexpression of Alg12p drives the addition of some
1,6-linked Man residue 6 in the absence of the central-arm
1,2-linked Man 10 (Scheme S1A), whose addition by Alg9p normally precedes it (Hubbard and Robbins, 1980
). What is particularly interesting about this observation is that the absence of
1,2-Man 9 on this Man7 glycan, characterized by HPLC size and
1,2-mannosidase sensitivity, suggests that Alg9p adds both residues 10 and 9 to wild-type OSL. Noteworthy in this regard, a candidate enzyme to add the last
1,2-Man to OSL (residue 9, Scheme S1A) has not been identified by genetic or homology searching methods.
In this study, addition of residue 6 in alg12 yeast with normal levels of Alg9p could not be detected. The J2,3 and J3,4 coupling constants of mannose residues in polysaccharides such as those studied here are ~3.5 Hz and ~10.0 Hz, respectively, giving rise to strong C2-H/C3-H 2D DQF COSY cross-peaks, allowing detection of trace amounts of such residues. The absence of any resonance for residue 4 at 4.15 (C2-H)/3.91 (C3-H) ppm (Winnik et al., 1982
) or that for 2-O-substituted residue 6 at 4.03 (C2-H)/3.96 (C3-H) ppm (Trimble and Atkinson, 1992
) verifies a paucity of 6. This means that Alg12p is required for addition of residue 6 in vivo, which suggests that under normal growth conditions yeast carefully regulate the level of the OSL mannosyltransferases to ensure ordered assembly of Glc3Man9GlcNAc2-PP-Dol (Burda et al., 1999
; Jakob et al., 1998
).
Glucosylation of Man5GlcNAc2-PP-Dol in the alg3 background is very low, with only ~7% of the chains transferred to protein containing the normal glucotriose unit (Verostek et al., 1993a). In contrast, alg9
yeast transfer a fully glucosylated Glc3Man6GlcNAc2 to protein, although little or no Glc3Man6GlcNAc2-PP-Dol accumulates in the OSL pool (Cipollo and Trimble, 2000
; Burda et al., 1999
). In some yeasts carrying the alg12 deletion, a small amount of Glc3Man7GlcNAc2-PP-Dol accumulates (Burda et al., 1999
), but it is clear from Figure 6 that Glc3Man7GlcNAc2 is the primary glycan transferred to protein in alg12
yeast. Overexpression of ALG6 in alg3
, alg9
, and alg12
yeasts increases the level of fully glucosylated OSL and, in the case of alg3
and alg9
, the level of glycosylation site occupancy on CPY (Burda et al., 1999
). This suggests that the addition of the first Glc residue by Alg6p is the rate-limiting step for full glucosylation, but that once fully glucosylated, even truncated manno-lipids can serve as good substrates for OST. Thus the enhanced glycosylation of invertase observed in alg12
cells relative to that seen in alg9
(Cipollo and Trimble, 2002
) is not due to increased OST function stimulated by the presence of the added
1,2-linked central-arm residue 10 but rather to that residues capacity to promote full glucosylation by potentiation of Alg6p activity.
Only a small residual amount of Glc remains on glycans from alg12 invertase, and it is clear from the [2-3H]Man pulse-chase study (Figure 6) that the glucotriose unit is both efficiently added to OSL and trimmed from glycoproteins in the ER. Alg9
cells also efficiently trim Glc residues and retain a similar amount of residue G1 to that seen in alg12
cells (Cipollo and Trimble, 2000
), as do wild-type cells on whole-cell N-glycans (Trimble and Atkinson, 1992
). This indicates that in the alg12
background, the upper arm residues 6 and 9 (Scheme S1) are not major structural determinants for the activities of either ER glucosidases I or II, nor does the retention of residue 10 in alg
impair glucosidase trimming.
Trimming in the ER of the glucotriose unit and 1,2-linked Man residue 10 (Scheme S1A) appears to act as a biological timer for protein maturation in yeast (Jakob et al., 1998
), as well as in higher eukaryotes (Helenius et al., 1997
; Chung et al., 2000
). Alg12
is compromised in its ability to remove misfolded CPY (designated CPY*) from the ER via ER-associated degradation, implying that failure to remove
1,2-linked Man 10 extends the time in which a misfolded protein is tolerated in the ER before degradation (Jakob et al., 1998
). It is noteworthy in the current work that over 85% of alg12
invertase glycans retained the central-arm
1,2-linked residue 10, demonstrating in vivo that the upper-arm
1,2Man
1,6Man- residues 6 and 9 (Scheme S1A) are required for optimum Mns1p activity. This confirms earlier in vitro studies that estimated the rate of residue 10 removal in Man7GlcNAc-ol structures lacking the upper-arm
1,2Man
1,6Man- residues 6 and 9 to be only 10% the rate of removal from Man9GlcNAc-ol (Ziegler and Trimble, 1991
).
Mannan outer-chain synthesis begins with the addition of 1,6-Man residue 12 to the lower-arm
1,3-Man core residue 5, catalyzed by Och1p (Reason et al., 1991
) in the cis-Golgi (Franzusoff and Schekman, 1989
; Scheme S1D). The substrate specificity of the Och1p was characterized in vitro using pyridylaminated oligosaccharides (Nakayama et al., 1997
). With the alg3 form of Man5GlcNAc2-PA (Scheme S1B without residue 7) as acceptor, only 9% as much Och1p product was formed as when Man89GlcNAc2-PA was the substrate. By contrast, 60% of a Man7GlcNAc2-PA, having the structure of the alg9
Man6GlcNAc (Scheme S1B) with upper-arm
1,6-Man residue 6 added (Scheme S1A), was elongated by Och1p. This defines core residues 6 and 7 (Scheme S1) as structural determinants for Och1p activity. Although the extent of elongation of Man8GlcNAc2-PA and Man9GlcNAc2-PA, which differ only by the presence of the central-arm
1,2-linked residue 10 in the latter (Scheme S1A), was similar, no kinetic data were reported in this study. Thus the rates of
1,6-Man addition to Man8 and Man9 processing intermediates may differ.
In this regard, Puccia and co-workers (1993) reported that [35S]-labeled invertase from mns1 cells migrated slightly faster on sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis than that of wild type. Furthermore, comparison alg3, alg9
, and alg12
glycans from invertase with one hexose added in the Golgi (Hex6GlcNAc2 for alg3, Verostek et al., 1993b
; Hex7GlcNAc2 for alg9
, Cipollo and Trimble, 2000
; and Hex8GlcNAc2 for alg12
, current study), shows that 3%, 22%, and 15%, respectively, have the
1,6-Man added by Ochlp. Thus in vivo the presence of residue 10 in both the mns1
and alg12
backgrounds appears to hinder Och1p activity to some extent. This is consistent with the observed mild hypoglycosylation of invertase seen in alg12
cells compared to wild-type cells (Cipollo and Trimble, 2002
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
N-glycans from alg12 external invertase
High-specific-activity external invertase was purified from alg12 cells harvested during glucose derepression under growth conditions optimized for low PrAp activity (Cipollo and Trimble, 2002
) as described (Verostek et al., 1993a
). N-linked oligosaccharides were hydrolyzed from invertase by treatment with PNGase F, isolated by solvent precipitation (Verostek et al., 2000
), and then chromatographed on a calibrated column of Bio-Gel P-4 (95 cm x 16 mm) with 0.1 N acetic acid/1% 1-butanol as the eluant at 8.8 ml/h at room temperature. Fractions of 0.73 ml were collected, and aliquots were assayed for total hexose and radioactivity from included internal marker(s) of Glc3[3H]Man9GlcNAc2 and/or [3H]Man3GlcNAc-ol.
Glycosidase digestions
Endo H and PNGase F digestions followed standard protocols (Tarentino et al., 1989). SDS was removed from the solubilized glycoproteins prior to PNGase F digestion by precipitation with 80% acetone and solubilization in 50 mM sodium phosphate buffer, pH 8.5 (Verostek et al., 2000
).
MS
MALDI/TOF MS was performed on a Bruker Reflex Instrument. Samples of 2550 pmol were prepared with 2,5-dihydroxybenzoic acid as matrix. Data accumulated for 1050 3-ns pulses of the 337-nm laser were averaged for each sample. Analyses were performed in linear and reflective mode.
HPAEC branch isomer analysis
Pooled aliquots of N-glycans were chromatographed on an HPAEC system using a voltage PAD response detector and an analytical (4 x 250 mm) PA-100 column. Samples were separated using 100 mM NaOH accompanied by the following sodium acetate gradient: isocratic at 35 mM for 5 min, and then 35170 mM over 45 min. Individual runs included raffinose or known glycans as internal standards.
1H NMR spectroscopy
Oligosaccharides (0.151.0 mg) were exchanged with D2O and examined at 300°K and/or 318°K by 1D and 2D DQF COSY phase-sensitive 1H NMR spectroscopy at 500 MHz as described (Cipollo and Trimble, 2000). Line broadening of 12 Hz/Hz was used in both dimensions of 2D DQF COSY experiments for signal enhancement, and a skewed sine-bell weighting function was used in t2 to reduce dispersive line shape.
[2-3H]Man pulse-chase analysis of N-glycan processing in vivo
Alg12 cells were grown overnight to stationary phase in YPD and collected by centrifugation for 5 min at 3000 rpm at room temperature in a Sorvall TC6 centrifuge equipped with an H400 rotor. The yeast were washed twice in glucose-freeYP medium by centrifugation and incubated in the presence or absence of 5 mM CST for 1.5 h in YP + 1% glucose. The yeast cells were again washed twice in glucose-free YP medium by centrifugation; cells (2 x 109) were resuspended to a total volume of 500 µl in YP + 0.15% Glc containing 500 µCi [2-3H]Man. After 2 min of labeling, a 2000-fold excess of unlabeled Man was added to the reactions. Aliquots of 125 µl were taken at 0, 1, and 10 min of chase, and reactions were terminated by rapid addition to 4 ml of CHCl3/CH3OH (3:2) while vortexing. The cell pellets were washed and OSL fraction isolated using the method of Zufferey et al. (1995)
). Labelled glycans were isolated from the pulse/chase cell pellets by endo H hydrolysis as described (Cipollo et al., 2001
).
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
2 To whom correspondence should be addressed; E-mail: trimble@wadsworth.org
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ballou, C.E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol., 185, 440470.[Medline]
Burda, P. and Aebi, M. (1998) The ALG10 locus of Saccharomyces cerevisiae encodes the -1, 2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation. Glycobiology, 8, 455462.
Burda, P. and Aebi, M. (1999) The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta, 1426, 239257.[ISI][Medline]
Burda, P., te Heesen, S., Brachat, A., Wach, A., Dusterhoft, A., and Aebi, M. (1996) Stepwise assembly of the lipid-linked oligosaccharide in the endoplasmic reticulum of Saccharomyces cerevisiae: identification of the ALG9 gene encoding a putative mannosyl transferase. Proc. Natl Acad. Sci. USA, 93, 71607165.
Burda, P., Jakob, C.A., Beinhauer, J., Hegemann, J.H., and Aebi, M. (1999) Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases. Glycobiology, 9, 617625.
Byrd, J.C., Tarentino, A.L., Maley, F., Atkinson, P.H., and Trimble, R.B. (1982) Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing. J. Biol. Chem., 257, 1465714666.
Cherry, J.M., Ball, C., Weng, S., Juvik, G., Schmidt, R., Adler, C., Dunn, B., Dwight, S., Riles, L., Mortimer, R.K., and Botstein, D. (1997) Genetic and physical maps of Saccharomyces cerevisiae. Nature, 387, 6773.[CrossRef][ISI][Medline]
Chung, D.H., Ohashi, K., Watanabe, M., Miyasaka, N., and Hirosawa, S. (2000) Mannose trimming targets mutant alpha(2)-plasmin inhibitor for degradation by the proteasome. J. Biol. Chem., 275, 49814987.
Cipollo, J.F. and Trimble, R.B. (2000) The accumulation of Man6GlcNAc2-PP-dolichol in the Saccharomyces cerevisiae alg9 mutant reveals a regulatory role for the Alg3p
1, 3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing. J. Biol. Chem., 275, 42674277.
Cipollo, J.F. and Trimble, R. B. (2002) Hypoglycosylation in the yeast alg12 mutant causes destabilization of PrA and proteolysis of external invertase. Glycobiology, 12 (in press).
Cipollo, J.F., Trimble, R.B., Chi, J.H., Yan, O., Lennarz, W.J., and Dean, N. (2001) The yeast ALG11 gene specifies addition of the terminal 1, 2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum. J. Biol. Chem., 276, 2182821840
Dean, N. (1995) Yeast glycosylation mutants are sensitive to aminoglycosides. Proc. Natl Acad. Sci. USA, 92, 12871291.[Abstract]
Franzusoff, A. (1992) Beauty and the yeast: compartmental organization of the yeast secretory pathway. Semin. Cell Biol., 3, 309324.[Medline]
Franzusoff, A. and Schekman, R. (1989) Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation. EMBO J., 8, 26952702.[Abstract]
Haeuw, F., Stecker, G., Wresiszeski , J., Monlreuil, J., and Michalske, J. (1991) Substrate specificity of rat liver cytosolic -D-mannosidase. Novel degradation pathway for oligomannoside type glycans. Eur. J. Biochem., 202, 12571268.[Abstract]
Hard, K., Mekking, A., Kamerling, J.P., Dacremont, G.A., and Vliegenthart, J.F. (1991) Different oligosaccharides accumulate in the brain and urine of a cat with alpha-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides. Glycoconj. J., 8, 1728.[ISI][Medline]
Helenius, A., Trombetta, E.S., Hebert, D.N., and Simons, J.F. (1997) Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol., 7, 193200.[CrossRef][ISI]
Hubbard, S.C. and Robbins, P.W. (1980) Synthesis of the N-linked oligosaccharides of glycoproteins. Assembly of the lipid-linked precursor oligosaccharide and its relation to protein synthesis in vivo. J. Biol. Chem., 255, 1178211793.
Huffaker, T.C. and Robbins, P.W. (1982) Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. J. Biol. Chem., 257, 32033210.
Huffaker, T.C. and Robbins, P.W. (1983) Yeast mutants deficient in protein glycosylation. Proc. Natl Acad. Sci. USA, 80, 74667470.[Abstract]
Jakob, C.A., Burda, P., Roth, J., and Aebi, M. (1998) Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J. Cell Biol., 142, 12231233.
Lussier, M., Sdicu, A-M., and Bussey, H. (1999) The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1426, 323334.[ISI][Medline]
Lussier, M., White, A-M., Sheraton, J., di Paolo, T., Treadwell, J., Southard, S.B., Horenstein, C.I., Chen-Weiner, J., Ram, A.F.J., Kapteyn, J.C., and others. (1997) Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics, 147, 435450.
Nakayama, K., Nokanistu-Shindo, Y., Tanaka, A, Haga-Toda, Y., and Jigami, Y. (1997) Substrate specificity of alpha-1, 6-mannosyltransferase that initiates N-linked mannose outer chain elongation in Saccharomyces cerevisiae. FEBS Lett., 412, 547550.[CrossRef][ISI][Medline]
Plummer, T.H. Jr. and Tarentino, A.L. (1991) Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum. Glycobiology, 1, 257263.[Abstract]
Puccia, R., Grondin, B., and Herscovics, A. (1993) Disruption of the processing -mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae. Biochem. J., 290, 2126.[ISI][Medline]
Reason, A.J., Dell, A., Romero, P.A., and Herscovics, A. (1991) Specificity of the mannosyltransferase which inintiates outer chain formation in Saccharomyces cerevisiae. Glycobiology, 1, 387391.[Abstract]
Reiss, G., te Heesen, S., Zimmerman, J., Robbins, P.W., and Aebi, M. (1996) Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway. Glycobiology, 6, 493498.[Abstract]
Stagljar, I., te Heesen, S., and Aebi, M (1994) New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus. Proc. Natl Acad. Sci. USA, 91, 59775981.[Abstract]
Tarentino, A.L., Trimble, R.B., and Plummer, T.H. Jr. (1989) Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Methods Cell Biol., 32, 111139.[ISI][Medline]
Trimble, R.B. and Atkinson, P.H. (1986) Structure of yeast external invertase Man814GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. J. Biol. Chem., 261, 98159824.
Trimble, R.B. and Atkinson, P.H. (1992) Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study. Glycobiology, 2, 5775.[Abstract]
Trimble, R.B., Byrd, J.C., and Maley, F. (1980) Effect of glucosylation of lipid intermediates on oligosaccharide transfer in solubilized microsomes from Saccharomyces cerevisiae. J. Biol. Chem., 255, 1189211895.
Trimble, R.B., Atkinson, P.H., Tschopp, J.F., Townsend, R.R., and Maley, F. (1991) Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. J. Biol. Chem., 266, 2280722817.
Trumbly, R.J., Robbins, P.W., Belfort, M., Ziegler, F.D., Maley, F., and Trimble, R.B. (1985) Amplified expression of Streptomyces endo-ß-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product. J. Biol. Chem., 260, 56835690.[Abstract]
Verostek, M.F., Atkinson, P.A., and Trimble, R.B. (1991) Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides. J. Biol. Chem., 266, 55475551.
Verostek, M.F., Atkinson, P.A., and Trimble, R.B. (1993a) Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum. J. Biol. Chem., 268, 1209512103.
Verostek, M.F., Atkinson, P.A., and Trimble, R.B. (1993b) Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. II. Structure of novel Man610GlcNAc2 Processing intermediates on secreted invertase. J. Biol. Chem., 268, 1210412115.
Verostek, M.F., Lubowski, C., and Trimble, R.B. (2000) Selective organic precipitation/extraction of released N-glycans following large-scale enzymatic deglycosylation of glycoproteins. Anal. Biochem., 278, 111122.[CrossRef][ISI][Medline]
Winnik, F.M., Carver, J.P., and Krepinsky, J.J (1982) Synthesis of model oligosaccharides of biological significance. 2. Synthesis of a tetramannoside and two lyxos-containing trisaccharides. J. Org. Chem., 47, 27012707.[ISI]
Ziegler, F.D. and Trimble, R.B. (1991) Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing -mannosidase. Glycobiology, 1, 605614.[Abstract]
Ziegler, F.D., Cavanagh, J., Lubowski, C. and Trimble, R.B. (1999) Novel Schizosaccharomyces pombe N-linked Man9GlcNAc isomers: role of the Golgi GMA12 galactosyltransferase in core glycan galactosylation. Glycobiology, 9, 497505.
Zufferey, R., Knauer, R., Burda, P., Stagljar, I., te Heesen, S., Lehle, L., and Aebi, M. (1995) STT3, a highly conserved protein required for yeast oligosaccharyltransferase activity in vivo. EMBO J., 14, 49494960.[Abstract]