Biochemistry Endocrinology and Metabolism Unit, Institute of Child Health At Great Ormond Street Hospital, University College London, 30 Guilford Street, London, WC1N 1EH, UK
Received on May 2, 2002; revised on July 22, 2002; accepted on July 26, 2002
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Abstract |
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Key words:
1-antitrypsin
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2D PAGE
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MALDI-TOF MS
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CDG
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Introduction |
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Comparison of the structures of glycosylated sites and unglycosylated sites has provided some information about the factors affecting the efficiency of glycosylation site occupancy (Hart et al., 1979; Bause and Hetkamp, 1979
). The proximity of the glycosylation sequon to the N-terminus and the nature of the amino acid before (Shakin-Eshleman et al., 1996
) and after (Mellquist et al., 1998
) the serine or threonine residue has been shown to affect the efficiency of glycosylation of a sequon. Many of these studies have been carried out in vitro or by overexpression of normal or mutated proteins in cells. Two other factors affect the pattern of glycosylation seen in a mature glycoprotein in vivo. They are the successful passage of the glycoprotein through the quality control mechanism in the ER and the half-life of the glycoprotein at its site of function.
The congenital disorders of glycosylation type 1 (CDG-I), which are characterized biochemically by underglycosylation of proteins, are an excellent model for studying the factors that affect the pattern of glycosylation of a mature glycoprotein (Keir et al., 1999; Aebi and Hennet, 2001
; Schachter, 2001
; Jaeken et al., 2001
). In these patients the glycosylation capacity is decreased because of defects in the assembly or transfer of the common dolichol-pyrophosphate-linked oligosaccharide precursor of asparagine-linked glycans. Consequently multiple forms (glycoforms) of serum/plasma glycoproteins with different degrees of glycosylation, that is, fully, partially, and nonglycosylated, appear in the plasma. It is assumed that under these conditions, only the more efficient sequons are glycosylated and that only those glycoforms with the appropriate structural features pass the quality control process and appear in the plasma. Previous analysis of serum transferrin from CDG-Ia patients has shown that the nonoccupancy of the two glycosylation sites in the underglycosylated form of transferrin is random (Wada et al., 1992
; Yamashita et al., 1993
; Henry et al., 1999
).
In this article, a strategy based on proteomic technology has been developed for investigating N-glycosylation site-occupancy in proteins separated by 2D polyacrylamide gel electrophoresis (PAGE). Using the new strategy, it has been found that the nonoccupancy of the three glycosylation sites in another plasma protein, 1-antitrypsin, from CDG-I patients is not random but determined by structural features. The implications of this observation for the intracellular transport and sorting of glycoproteins are discussed.
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Results |
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To confirm this conclusion, 1-antitrypsin isoforms iv were deglycosylated with PNGase F prior to tryptic digestion. The resultant peptide maps contained four masses, 1756.9 m/z, 3181.6 m/z, 3198.6, and 3691.8 m/z, that were not observed in the analysis of the M series. The masses 3181.6 m/z and 3198.6 m/z (Figure 3b) correspond to the amino acid sequence 4069 with oxidized methionine with and without pyroglutamate, respectively, but with aspartic acid at residue 46, indicating that this residue was glycosylated. Similarly the presence of mass 1756.9 (Figure 5b) indicated that asparagine 247 was also glycosylated. The presence of mass 3691.8 m/z, corresponding to amino acid sequence 70101 with an asparagine at position 83, confirmed that asparagine 83 was unglycosylated in all of the diglycosylated
1-antitrypsin isoforms in CDG-I. No apparent changes in the isotopic ratio of the peptides were observed in the mass spectra after the enzymic removal of the glycans using PNGase F, which converts an asparagine to aspartic acid with an increase in m/z of 1. This indicated very strongly that not even small amounts of asparagine 83 were glycosylated.
Analysis by MALDI-TOF MS of the glycans removed from isoforms (i)(v) was only possible for the more abundant diglycosylated species (ii) and (iii) (Figure 1). The spectrum for the glycans from isoform (ii) contained two ions of masses of 2223.4 m/z and 2880.1 m/z, corresponding to biantennary and triantennary complex glycans. The carbohydrate content of isoform (ii), combined with the peptide data showing both the presence of the N-terminal amino acids and the absence of a glycan structure at asparagine 83, indicates that this isoform is the diglycosylated equivalent of the M4 isoform of 1-antitrypsin (Figure 1). This is consistent with the mass decrease of approximately 2.2 kDa and the cathodal shift in the pI of 0.1 pH units observed by 2D PAGE.
The MS analysis of the glycans released from isoform (iii) contained only one major ion species of mass 2223.4 m/z, corresponding to a biantennary complex glycan (Figure 6). The glycan and peptide mass spectral data indicate that isoform (iv) is the diglycosylated equivalent of the M6 isoform of 1-antitrypsin, specifically lacking a biantennary glycan at asparagine 83. This is consistent with the pI and Mr coordinates observed for isoform (iii) on 2D PAGE (Table I).
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Isoform (i), which was only detected in CDG-Ic, has the same pI as isoform M6, which possesses three biantennary glycans. Therefore, as isoform (i) retains the N-terminal sequence, it probably has two triantennary complex glycans at asparagines 46 and 247. Thus in all the diglycosylated isoforms of 1-antitrypsin in plasma, in all the forms of CDG-I, asparagine 83 is preferentially not glycosylated.
Analysis of the monoglycosylated 1-antitrypsin isoform (isoform vi)
MS analysis of the peptides liberated from spot (vi) identified this protein as 1-antitrypsin. The detection of the reporter peptide for the N-terminus with a mass of 2819.2 m/z confirmed that the N-terminus was present. The presence of the mass of 3691.8 m/z, which is diagnostic for amino acids 70101 with an asparagine at position 83, confirmed that this asparagine was also unglycosylated in this isoform (Figure 4c). A mass of 1755.9 m/z, which is due to amino acids 244259 with an asparagine at position 247, was also detected (Figure 5c). This showed that the second unoccupied glycosylation site was asparagine 246. The masses corresponding to an unglycosylated asparagine 46 (see Table II) were not present in the spectrum. These results indicated that glycosylation sites 83 and 247 were not occupied in this isoform of
1-antitrypsin.
This was confirmed by the MS analysis of the tryptic digestion of deglycosylated component (vi) (Figure 3b). The appearance of two ions of masses 3181.6 m/z and 3198.6 m/z, corresponding to the amino acid sequence 4069 and its oxidized methionine derivative with both containing an aspartic acid, indicated that asparagine 46 was glycosylated. The diagnostic reporter masses for unglycosylated asparagines, 83 and 247, were also detected. Therefore, isoform (vi) is monoglycosylated at asparagine 46 and retains the first five amino acids.
It was not possible to analyze the glycans attached to asparagine 46 in isoform (vi) by in-gel digestion but theoretically a bi- or a triantennary glycan could be present. The estimated pI from its position on the 2D gel, 5.26, is consistent with the presence of two sialic acids, that is, a biantennary glycan. The fully defined structure, M6, which has three biantennary glycans and the N-terminal sequence, has a pI of 5.05. The pI of isoform (vi) is consistent with the loss of two biantennary glycans, which would increase the pI by approximately 0.2 pH units. Therefore, isoform (vi) is probably the monoglycosylated equivalent of M6 with only one biantennary glycan at asparagine 46.
Analysis of the nonglycosylated 1-antitrypsin isoform (isoform vii)
MS analysis of the peptides liberated from spot (vii) identified this protein as an isoform of 1-antitrypsin with an intact N-terminus. The presence of the peptide masses of 3180.6/3197.6 m/z (Figure 3c), 3691.8 m/z (Figure 4c), and 1755.9 m/z (Figure 5c) (see Table II) indicated that asparagines 46, 83, and 247, respectively, were all unglycosylated in isoform (vii) of
1-antitrypsin.
Treatment of isoform (vii) with N-glycanase, prior to tryptic digestion, did not produce any additional masses and confirmed that this was the nonglycosylated isoform of 1-antitrypsin. Theoretically, on our 2D PAGE system, the nonglycosylated isoform of
1-antitrypsin with an intact N-terminus should have a pI of approximately 5.35 U, which is in good agreement with the estimated observed pI of 5.36. The proposed macro- and microheterogeneity of the underglycosylated isoforms of
1-antitrypsin in CDG-I patients are shown in Figure 7.
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Discussion |
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The pattern of occupation of N-glycosylation sites in the 1-antitrypsin isoforms present in plasma will depend on several factors. First, the initial degree of glycosylation of the protein will depend upon the glycosylation capacity of the hepatocytes and the integrity of the nascent polypeptide chain. Because the structural gene for
1-antitrypsin is normal in CDG-I, the appearance of underglycosylated isoforms in the plasma must be due to the decreased capacity for glycosylation.
Second, the flux of fully and partially glycosylated 1-antitrypsin through the lumen of the rough ER to the sites of N-linked glycan processing will be determined by the editing/quality control mechanism, mediated by calnexin and other chaperones (Molinari and Helenius, 1995; Choudhury et al., 1997
). It has been shown that the correct folding of
1-antitrypsin during its synthesis involves a cyclic association with and release from calnexin until it is correctly folded (Choudhury et al., 1997
). This process is regulated by repeated glucosylation and deglucosylation, which is mediated by the enzymes UDP-glucose:glycoprotein glucosyltransferase and glucosidase II. Incorrectly folded isoforms of
1-antitrypsin are targeted to the proteasome for degradation by ubiquitination of the calnexin (Qu et al., 1996
). The overall decrease in the plasma concentration of
1-antitrypsin in CDG-I (Jaeken et al., 2001
) suggests that a significant proportion of the
1-antitrypsin is rejected by this mechanism. The fully glycosylated isoforms constitute about 6070% of the total plasma
1-antitrypsin in CDG-I (Henry et al., 1999
) (equivalent to
40% of the normal concentration) suggesting that underglycosylated isoforms are not exported efficiently. As unglycosylated recombinant
1-antitrypsin has been shown to be active in vitro (Rosenberg et al., 1984
; Travis et al., 1985
), it is probable that the small amount of unglycosylated
1-antitrypsin present in plasma from CDG-I patients is also functional. This suggests that either
1-antitrypsin can fold up correctly independently of glycosylation by an alternative chaperone-mediated pathway (Molinari and Helenius, 2000
) or some
1-antitrypsin bypasses the calnexin/calreticulin editing mechanism (Cooper et al., 1997
).
The next cause of heterogeneity is the processing of the N-linked glycans, which takes place predominantly in the Golgi apparatus. The glycans on the plasma isoforms of 1-antitrypsin are fully processed in CDG-I, as we have shown here (Figure 6) and previously (Mills et al., 2001a
,b
). In fact there is increased fucosylation and branching of the glycans on both the fully and partially glycosylated isoforms in CDG-I (Mills et al., 2001b
). This is particularly marked in CDG-Ic, which probably explains why isoform (i), which has two triantennary complex glycans, is only detected in CDG-Ic (Figure 1). The posttranslational removal of the five N-terminal amino acids also takes place in the Golgi. This modification appears to be unaffected by underglycosylation, as judged by the appearance of isoforms (iv) and (v) in the plasma of CDG-I patients.
The final factor affecting the pattern of isoforms of 1-antitrypsin in plasma is their clearance from the circulation. The absence or decrease in carbohydrate might be expected to lower the stability of an isoform, but, in contrast, it diminishes removal from circulation by carbohydrate-mediated endocytosis. Unglycosylated recombinant human
1-antitrypsin has been demonstrated previously as having a shorter half-life in rabbits than the normal glycosylated form (Travis et al., 1985
).
The pattern of N-glycosylation site occupancy observed in the mature forms of 1-antitrypsin in plasma could represent the pattern of glycosylation after synthesis or the spectrum of glycoforms allowed through the editing/quality control mechanism. If it were the latter, glycosylation of asparagine 46 would be a minimal requirement for clearance of
1-antitrypsin by the editing/quality control mechanism, except for the small amount of unglycosy- lated
1-antitrypsin. Underglycosylated but functional
1-antitrypsin is present in the plasma of healthy individuals heterozygous for the (PIZBristol) variant, in which genetic abrogation of the asparagine 83 glycosylation sequon occurs (Mills et al., 2001a
; Lovegrove et al., 1997
). This N-glycosylation mutant leads to a mild deficiency of plasma
1-antitrypsin but is not thought to be disease-causing.
1-Antitrypsin mutagenized at asparagine 83 is secreted less efficiently from COS-1 cells but is active (Samandari and Brown, 1993
), confirming that glycosylation of asparagine 83 is not essential for secretion. Diglycosylated
1-antitrypsin with glycans attached at asparagines 83 and 247 or 46 and 83, or monoglycosylated
1-antitrypsin with a single glycan at asparagine 83 or 247, were not detected in plasma. These forms were therefore either removed by the editing/quality control mechanism or not synthesized in the first place.
If certain isoforms were not synthesized at all, then the pattern of glycosylation is very specific, with asparagine 46 being glycosylated most efficiently, followed by asparagine 247 and finally asparagine 83. The preferential occupation of the glycosylation site closest to the N-terminal is consistent with other data on the positional importance of glycosylation in the folding and maturation of glycoproteins (Gavel and von Heijne, 1990; Branza-Nichita et al., 2000
). It is interesting to speculate on the molecular basis for this pattern of glycosylation. Sequons containing threonine rather than serine as the third amino acid in the sequon have been reported to be glycosylated more efficiently in vitro (Shakin-Eshleman et al., 1996
; Mellquist et al., 1998
). However, all three glycosylation sites in
1-antitrypsin have threonine in the third position of the glycosylation sequon.
The middle amino acid of the Asn.X.Ser/Thr sequence has also been shown to affect the efficiency of glycosylation in the rabies glycoprotein when expressed in vitro (Shakin-Eshleman et al., 1996). The Asn 46 in
1-antitrypsin is followed by a serine, which was the most efficient X residue in the viral glycoprotein experiments. Asn 247 precedes an alanine, which is also an efficient amino acid (Shakin-Eshleman et al., 1996
), whereas Asn 83 is followed by leucine, which was one of the least efficient amino acids when serine was the third amino acid. The amino acid following the sequon is also an important determinant of glycosylation efficiency in the viral glycoprotein expression system, especially if the third amino acid is serine (Mellquist et al., 1998
). However the amino acids following the three sequons in
1-antitrypsin, asparagine, glutamic acid, and alanine, respectively, have comparable effects on the glycosylation of threonine-containing sequons. Therefore it is possible that asparagine 83 is glycosylated less efficiently under conditions of decreased glycosylation capacity because the X residue in the sequon is leucine.
The efficiency of glycosylation of a sequon in DNase I has been shown to depend on the X amino acid and the tissue of origin (Nishikawa and Mizuno, 2001). It is probable that the major factor determining the pattern of underglycosylation of
1-antitrypsin under conditions of inadequate glycosylation capacity, as in CDG-I, is the efficiency of glycosylation of sequons. Subsequently any underglycosylated
1-antitrypsin isoforms that cannot fold properly will be removed by the quality control mechanism in the ER. The half-lives of the isoforms secreted into the blood in circulation will also be affected by their state of glycosylation. Therefore the pattern of
1-antitrypsin isoforms found in plasma will be a reflection of all these processes.
It is probable, according to previously published data, that the sequon closest to the N-terminal of a protein will generally be glycosylated. However, the occupancy of other sequons will depend upon the efficiency of glycosylation of each sequon in that particular protein. Therefore the pattern of occupancy of N-glycosylation sites of plasma glycoproteins in CDG-I will vary from protein to protein. The same factors probably determine the occupancy of glycosylation sites under conditions in which glycosylation capacity is not restricted, together with the physiological state and type of cell in which the protein is synthesized.
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Materials and methods |
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Material from CDG-I patients
Plasma was obtained from patients who had been diagnosed with CDG-I on the basis of an abnormal isoelectric focusing profile for transferrin, an enzymic deficiency or DNA analysis (Keir et al., 1999). Two patients had CDG-Ia with a deficiency of phosphomannomutase and another had CDG-Ic due to a deficiency of dolichyl-P-Glc: Man9GlcNAc2-PP-dolichyl
1,3-glucosyl-transferase deficiency (hALG6). The enzymic defect in the fourth patient was unknown, but his plasma glycoproteins had the characteristic underglycosylation of CDG-I. Analysis of the glycans released from the plasma glycoproteins confirmed the absence of a defect in the processing of the carbohydrate chains and excluded CDG-II (Mills et al., 2001a
) (data not shown).
2D PAGE
All 2D PAGE analyses were performed according to Mills et al. (2001a) with minor modifications. Plasma samples for both analytical (2.5 µl) and preparative analyses (10 µl for MALDI-TOF MS analysis) were focused on Immobiline DryStrips (18 cm, pH 4.55.5 and pH 47) using a LKB-Multiphor II focusing unit (Amersham Biosciences). Isoelectric focusing was carried out for a minimum of 75 kV h and a maximum of 100 kV h. Focused strips were snap-frozen in liquid nitrogen and stored at -80°C until required.
Analytical gels consisted of acrylamide 10% (w/v) with piperazine diacrylamide 1.6% (w/v) cross-linker. Preparative gels were prepared using acrylamide 10% (w/v) with bis-acrylamide 0.1% (w/v) or 1.0% for subsequent in-gel digestions with PNGase F and trypsin, respectively. Resolubilization of the focused proteins and carboamidomethylation of cysteine residues were performed according to Diettrich et al. (1988). Sodium dodecyl sulfatePAGE was carried out in a Protean II Multicell electrophoresis unit (BioRad).
Analytical gels were silver-stained using an automatic gel stainer (Amersham Biosciences) as described by Hochstrasser et al. (1988) and the preparative gels were silver-stained according to Shevchenko et al. (1996)
. Proteins separated by 2D PAGE were identified by mass mapping studies (see later discussion). The pI and molecular weight coordinates for the 2D PAGE system were determined by comparison of the identified proteins with the Swiss-Prot on-line plasma 2D PAGE database (Geneva, Switzerland, available online at www.expasy.ch/cgi-bin/map2/def?plasma_human).
In-gel proteolytic digestion and extraction of peptides and glycans
In-gel tryptic digestions were performed as described by Shevchenko et al. (1996). The released peptides were extracted from the gel (Mills et al., 2001a
) and desalted on a C-18 stationary phase micro-column, as described by Mills et al. (2000)
. The desalted peptide solution was dried by centrifugal evaporation and reconstituted in 10 µl of 0.1% trifluoracetic acid for MS. N-linked glycans were released by in-gel digestion with PNGase F according to Kuster et al. (1998)
and Mills et al. (2001b)
.
MALDI-TOF MS
MS was carried out on a MALDI-TOF instrument, fitted with a reflectron and a 337 nm UV laser (TOF Spec E, MicroMass, Manchester, UK). Peptide analyses were performed in positive ion mode with the following voltages: source 20 kV, extraction 19.95 kV, focus 16.5 kV, and reflectron 25 kV. Spectra were acquired by averaging over a period of five scans of highest signal. Data were acquired in reflectron mode, operating over a mass range of 6000 m/z with matrix suppression set at 650 mass units. Peptides were analyzed using an cyano-4-hydroxycinnamic acid/fucose co-matrix as described by Mills et al. (2000)
. Glycans were analyzed by MALDI-TOF MS operating in negative linear mode according to Mills et al. (2001b)
using trihydroxyacetophenone:ammonium citrate matrices as described by Papac et al. (1996)
.
Data analysis was carried out using MassLynx and BioLynx data analysis software, Protein Prospector database software at University of San Francisco (http://falcon.ludwig.ucl.ac.uk/mshome3.2.htm), and PAWS proteomic analysis software.
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Acknowledgements |
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: b.winchester{at}ich.ucl.ac.uk
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Abbreviations |
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References |
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