Glycobiology Advance Access originally published online on September 23, 2007
Glycobiology 2007 17(12):1388-1403; doi:10.1093/glycob/cwm097
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Identification of N-Glycosylated Proteins from the Central Nervous System of Drosophila Melanogaster
2 Department of Biochemistry and Biophysics, 77843-2128, Texas A&M University, College Station, TX, USA
3 Complex Carbohydrate Research Center and Department of Chemistry, University of Georgia, Athens, GA 30602, USA
4 Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
1 To whom correspondence should be addressed: Tel: +979-458-4630; Fax: +979-845-9274; e-mail: panin{at}tamu.edu; Tel: +706-542-7806; Fax: +706-542-4412; e-mail: Iwells{at}ccrc.uga.edu; Tel: +706-542-2740; Fax: +706-542-4412; e-mail: myiemeyer{at}ccrc.uga.edu
Received on July 20, 2007; revised on August 28, 2007; accepted on August 31, 2007
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Abstract |
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Although the function of many glycoproteins in the nervous system of fruit flies is well understood, information about the glycosylation profile and glycan attachment sites for such proteins is scarce. In order to fill this gap and to facilitate the analysis of N-linked glycosylation in the nervous system, we have performed an extensive survey of membrane-associated glycoproteins and their N-glycosylation sites isolated from the adult Drosophila brain. Following subcellular fractionation and trypsin digestion, we used different lectin affinity chromatography steps to isolate N-glycosylated glycopeptides. We identified a total of 205 glycoproteins carrying N-linked glycans and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. Glycan profiles were analyzed separately for glycopeptides that were strongly and weakly bound to Concanavalin A (Con A), or that failed to bind Concanavalin A, but did bind to wheat germ agglutinin (WGA). High- or paucimannosidic glycans dominated each of the profiles, although the wheat germ agglutinin-bound glycan population was enriched in more extensively processed structures. A sialylated glycan structure was unambiguously detected in the wheat germ agglutinin-bound fraction. Despite the large amount of starting material, insufficient amount of glycopeptides was retained by the Wisteria floribunda (WFA) and Sambucus nigra columns to allow glycan or glycoprotein identification, providing further evidence that the vast majority of glycoproteins in the adult Drosophila brain carry primarily high-mannose, paucimannose, and hybrid glycans. The obtained results should facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the central nervous system (CNS) of Drosophila.
Key words: CNS / Drosophila glycoproteomics / lectin chromatography / mass spectrometry / N-glycosylation
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Introduction |
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In higher organisms, glycosylation plays important roles in numerous developmental and physiological processes. It is one of the most common posttranslational modifications of proteins that is often evolutionary conserved and, at the same time, least understood from both structural and functional perspectives. Cell adhesion, cell signaling, self-non-self recognition, subcellular and extracellular trafficking, receptor modulation, and activation, all these crucial biological processes have been shown to be affected by glycosylation (Ohtsubo and Marth 2006
).
N-linked glycosylation is the best understood type of carbohydrate modification. One of the most widely known functions of N-glycosylation is in protein translocation, folding, and secretion (Helenius and Aebi 2004; Chavan and Lennarz 2006), while the glycans themselves may not necessarily be required for the protein's function. In other cases, N-glycosylation has a profound effect on the protein's biological activity via influencing protein conformation and stability (Blum et al. 1993; Wyss et al. 1995; Wormald and Dwek 1999), protein–protein interactions (Purohit et al. 1997), or protecting against proteolytic cleavage (Wittwer and Howard 1990). Despite the fact that the structure and biosynthetic pathway of N-linked glycans is relatively well studied, it is still very difficult to predict which potential N-glycosylation sites will be used, what type of glycans will be attached at these sites, and which of the many potential functions these glycans will serve (Ohtsubo and Marth 2006).
Glycosylation in the nervous system is thought to play a particularly important and dynamic role in both vertebrates and invertebrates given the extraordinary complexity of synaptic connections and different specialized brain structures (Kleene and Schachner 2004). The highly dynamic expression pattern of different glycan structures reflects their specific requirement during distinct developmental periods, as illustrated in the leech nervous system (Tai and Zipser 2002) or in mice (Henion et al. 2005; Weinhold et al. 2005). Because of the complexity of the nervous system and glycosylation pathways in vertebrates, invertebrate organisms provide a unique opportunity to study the evolutionary conserved aspects of glycosylation at a simplified level (Altmann et al. 2001). Drosophila represents an especially attractive model system because of its well-characterized development and well-known amenability to genetic manipulation (Adams and Sekelsky 2002).
In order to address more systematically the different functions of N-glycans (e.g., affecting protein solubility versus being involved in protein–protein interactions), we would need to know what proteins are glycosylated and catalogue which of their potential glycosylation sites are utilized, and what glycan structures occupy those sites. This in turn would allow further studies to be performed that would address the role of glycans at these specific N-glycosylation sites in vivo using molecular and genetic approaches.
With the recent marked developments in the sensitivity of mass spectrometric methods for proteomic and glycomic analyses, such system-wide surveys are becoming feasible. Examples of such endeavors are the recent identification of a large number of glycoproteins and their N-glycosylation sites from Caenorhabditis elegans (Kaji et al. 2003; Fan et al. 2005) and glycomic studies in Drosophila (Leonard et al. 2006; North et al. 2006; Rendic et al. 2006; Aoki et al. 2007).
We have in the past determined the in vitro enzymatic activity and acceptor specificity of the first insect sialyltransferase, DSiaT (Koles et al. 2004). As in vitro acceptors, this enzyme prefers terminal LacdiNAc (GalNAcß1,4GlcNAc) and LacNAc (Galß1,4GlcNAc) residues found on N-linked glycans of glycoproteins. We also found that Drosophila sialyltransferase is specifically expressed in the central nervous system (CNS) throughout all developmental stages. This gave us the initial incentive to characterize N-linked glycosylation in Drosophila brain with particular focus on potentially sialylated membrane glycoproteins. In general, information about glycosylation in the Drosophila CNS is rather limited (Seppo and Tiemeyer 2000; Fabini et al. 2001; Wilson 2002; Leonard et al. 2006; Rendic et al. 2006; Sarkar et al. 2006). Drosophila neural tissue is enriched for a class of N-linked glycans collectively known as HRP-epitopes, consisting primarily of paucimannosidic cores that carry 
3- and 6-linked fucose on their reducing terminal GlcNAc residues (Jan and Jan 1982; Snow et al. 1987). At the same time, the sialylated structures previously identified in Drosophila embryos do not undergo core fucosylation and, therefore, are not HRP-epitope glycans (Aoki et al. 2007).
In this study, we have set out to use lectin affinity chromatography with agarose-conjugated Concanavalin A (Con A), wheat germ agglutinin (WGA), Sambucus nigra (SNA), and Wisteria floribunda (WFA) lectins to enrich for N-glycosylated glycoproteins from the membrane fraction of adult heads of Drosophila melanogaster. The tryptic digest fraction of membrane glycoproteins was applied to a series of these lectin affinity columns. Given that the N-linked glycans of Drosophila are characterized by the predominance of oligo- and paucimannosidic structures with a relatively small fraction of hybrid and complex structures (Leonard et al. 2006; North et al. 2006; Rendic et al. 2006; Aoki et al. 2007), the Con A and WGA affinity chromatography steps can be considered as an enrichment step for the majority of N-glycosylated glycopeptides. Further subfractionation according to the terminal sugar residues was attempted using SNA and WFA lectins. To mark occupied glycosylation sites on fractionated glycopeptides, a stable isotopic mass shift was introduced at glycosylated asparagine residues by digesting glycopeptides with PNGaseF (N-glycanase) in H218O-water. Glycoproteins bearing HRP-epitopes have been previously analyzed (Desai et al. 1994; Wang et al. 1994; Sun and Salvaterra 1995). Thus, digestion with PNGaseF, which is unable to remove N-linked glycans bearing 3-linked Fuc, focused our analysis on glycopeptides that do not bear HRP-epitopes. We anticipated that this approach might enhance our ability to detect glycopeptides bearing complex, possibly sialylated glycans. In the end, released glycan profiles and glycopeptide sequences were successfully determined from the Con A and WGA bound fractions. A sialic acid-containing structure was unambiguously detected in the WGA fraction. We identified a total of 205 unique glycoproteins and revealed their 307 utilized N-glycosylation sites. We then used this information to analyze possible biases in amino acid distribution around the N-glycosylation site.
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Results |
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Lectin fractionation of Drosophila head membrane glycopeptides
To identify N-glycosylated glycoproteins from the CNS of Drosophila, including potentially sialylated structures, we have used adult heads as a starting material for the isolation of membrane-associated glycoproteins (Figure 1A). In order to obtain sufficient material for further analysis, we scaled up our typical fly cultures by growing the flies in larger containers. Within about a year, we collected enough flies to yield 50 g of heads (one fly head is approximately 0.1 mg, thus approximately half a million flies were used for this study). Initially, we tried to use SNA lectin-affinity chromatography (Shibuya et al. 1987
) to capture 2-6 sialylated glycopeptides from the unfractionated complex mixture of tryptic peptides and glycopeptides obtained from total membrane-associated protein extract from Drosophila heads. However, this approach failed to recover any glycopeptides (data not shown), probably due to the extremely low amount of these structures relative to the high background "noise" of nonspecific interactions with peptide and irrelevant glycopeptide. Thus, we decided to use Con A and WGA chromatography as an enrichment step for N-glycosylated glycopeptides (Figure 1B) to decrease the background (from non- or O-glycosylated peptides) and to increase the concentration of potentially sialylated glycopeptides.
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Con A is known to bind oligomannosidic structures with high affinity, and to weakly retain hybrid and complex biantennary, but not triantennary structures (Krusius et al. 1976
). In the light of available data from Drosophila embryos (North et al. 2006; Aoki et al. 2007), larvae (Williams et al. 1991), adults (Fabini et al. 2001), and cultured cells (Aumiller and Jarvis 2002; Rendic et al. 2006), it is expected that complex structures not bound by Con A (such as triantennary glycans) will be present in the extract in only minute amounts. Hence, we expected that Con A chromatography should capture the vast majority of N-glycosylated glycopeptides, including potentially sialylated glycopeptides (Krusius et al. 1976). In addition to SNA chromatography, we also used WFA lectin to trap glycopeptides with terminal GalNAc structures (Piller et al. 1990). We were interested in the WFA-bound fraction since it should include glycans with potential LacdiNAc termini, which were shown to be the preferred acceptors for Drosophila sialyltransferase (Koles et al. 2004). Finally, we have also included a WGA-affinity step to broaden the range of captured glycopeptides in addition to those bound by Con A, especially since WGA shows high affinity for certain GlcNAc and NeuAc residues (Yamamoto et al. 1981). In the end, the combination of these lectin affinity steps yielded seven fractions (Figure 1B): V1 – Con A weakly bound and not bound by SNA and WFA; V2 – Con A strongly bound, not bound by SNA and WFA; V3 – Con A weakly bound, WFA-bound, not bound by SNA; V4 – Con A weakly bound, SNA-bound; V5 – Con A strongly bound, not bound by SNA and bound by WFA; V6 – Con A strongly bound, SNA-bound; and V7 – not bound by Con A and bound by WGA. Only V1, V2, and V7 fractions were amenable to further glycan analysis and peptide sequencing (Figure 1), while no data could be obtained from V3–V6 fractions due to the low amount of the recovered material.
Carbohydrate analysis of the recovered glycopeptide fractions
Glycans released from the glycopeptides of V1, V2, and V7 by PNGaseF were predominantly of the high-mannose or paucimannose type (Figure 2). These profiles are consistent with previous analyses demonstrating the high prevalence of such glycans in Drosophila (Williams et al. 1991; Leonard et al. 2006; North et al. 2006; Aoki et al. 2007). The ratio of more processed glycans (NM3N2F, M3N2F, M3N2, and M2N2F, see Figure 3 for structures and nomenclature) relative to high-mannose glycans was significantly higher in the WGA-bound fraction (V7) (Figure 3). Minor, more extensively processed glycans were also detected by Total Ion Mapping (TIM) and by NSI-MSn. In particular, fragment ions consistent with the presence of a single, sialylated glycan (SA-GalNM3N2, m/z = 1983) were detected in V1, V2, and V7. However, the MSn signal intensities for signature fragments of SA-GalNM3N2 were weak and the fragmentation profiles were incomplete for V1 and V2. Fragmentation of the SA-GalNM3N2 precursor ion at m/z = 1983 was most complete and of the highest intensity in the V7 fraction (Figure 4). The fragment ions detected by MS2 and MS3 at m/z = 1835, 1330, 1128, 881, and 676 are entirely consistent with the indicated structure and with the previously published characterization of this glycan in Drosophila embryos (Aoki et al. 2007). The enhanced detection of the sialylated glycan as well as the increased ratio of processed glycans (NM3N2F, M3N2F, M3N2, and M2N2F) relative to high-mannose glycans in V7 (Figure 3), indicate that the WGA column has enriched the glycopeptide preparation for peptides bearing more extensively processed N-linked oligosaccharides. This enrichment is likely explained by partial depletion of oligomannose structures in the preceding Con A chromatography step (Figure 1) as well as by the preferential affinity of WGA for GlcNAc residues and the chitobiose core of N-linked oligosaccharides (Yamamoto et al. 1981) which are probably more easily accessible in the extensively processed N-linked oligosaccharides in comparison to mannose-decorated unprocessed structures.
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Peptide and N-glycosylation site analysis of the glycopeptide fractions
The enriched peptides were deglycosylated via PNGaseF in H218O to tag the site of N-linked attachment (Kaji et al. 2003
). The resulting peptides were analyzed by LC-MS/MS. A total of 291 peptide sequences with 307 utilized glycosylation sites were determined from V1, V2, and V7 fractions representing Con A- and WGA-bound glycopeptides. These peptides correspond to 205 different glycoprotein-encoding genes (Table I, Figure 4). Cell adhesion molecules (12%) represent the largest fraction of identified proteins with known molecular functions, followed by transporters (10%), cell surface receptors (9%), various enzymes (at 8% each), and ion channel molecules (5%) (Table I, Figure 5). Many of the identified proteins are known to be highly enriched in the nervous system, such as the cell adhesion molecules neuroglian, chaoptin, rhodopsin, DSCAM, indicating that they are among the most abundant glycosylated proteins within the brain. Only a small fraction of identified glycoproteins represent extracellular matrix components, suggesting that the extracellular matrix did not significantly contribute to the membrane fraction that we used for the analysis (Materials and methods).
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N-Glycosylation site utilization in Drosophila brain glycoproteins
Using the identified glycoprotein sequences and their mapped N-glycosylation sites, we have also examined the distribution of amino acids in the vicinity of the N-glycan attachment site (+/–20 residues upstream and downstream from the asparagine of the experimentally verified glycosylated consensus sequon, N-X-S/T). Within this region, we have compared the experimentally obtained distributions of different amino acids at each sequence position to the distributions expected in a random Drosophila peptide sequence (see Materials and methods). The comparison is summarized as a chart of biases, where we plotted only those amino acids whose distributions were significantly different (more than 2 SD away) from the ones expected for their appearance in a random sequence (Figure 6). The analysis was performed in two ways: (a) we analyzed the occurrence of individual amino acids or small groups of amino acids in the vicinity of the glycosylation site (Figure 6A); and (b) the analysis was also carried out for five groups of amino acids according to their physicochemical properties (Petrescu et al. 2004
; Nelson and Cox 2005) (Figure 6B). The analysis of individual/small groups of amino acids indicates a bias for the presence of glycine and threonine at positions +1 and +4, respectively. At the same time, serine was disfavored at position +3, and the frequency of prolines at positions just upstream of the glycosylation site (–3, –2, and –1) was reduced (Figure 6A). The analysis of grouped amino acids revealed: (i) an overall significant increase in the frequency of aromatic amino acids in the entire region analyzed around the N-glycosylation site, with several prominent positive biases upstream and downstream of the site; (ii) a strong preference for hydrophobic amino acids immediately following the glycosylation site (positions +1, +3); (iii) a significant negative bias against hydrophilic polar amino acids (–1, +3) and basic amino acids (+1, +4) around the sequon; (iv) a significant preference for positively charged amino acids at the –3 position (Figure 6B).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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In the present study, we have identified 307 glycosylation sites on 291 glycopeptides corresponding to 205 different glycoproteins from adult Drosophila heads and determined their utilized N-glycosylation sites. These results offer a remarkably rich source of information that will allow more detailed questions to be asked about the specific roles of N-glycans in the CNS. For example, the utilization of N-glycosylation sites of glycoproteins from the adult CNS can be compared with the glycosylation sites of the same proteins from other tissues and/or at different developmental stages, or with the N-glycosylation sites of orthologous proteins in other organisms.
Some general parallels to the nematode C. elegans Con A binding glycoproteome are apparent upon comparing the major classes of glycoproteins recovered (Fan et al. 2005). Cell adhesion molecules and diverse enzymes represent the largest functional group of known proteins identified in both cases, indicating the requirement for N-glycosylation of these glycoproteins. However, the largest group of identified glycoproteins comprises molecules with unknown functions, suggesting a considerable repertoire of unexplored glycoproteins expressed in the Drosophila CNS (Table I).
One interesting example of the glycoproteins identified in the present study, oligosaccharyltransferase 3 (OstStt3), is a subunit of the oligosaccharyltransferase (OT) complex. Intriguingly, the function of N-glycosylation sites on this particular subunit has recently been addressed in yeast, where the different components of OT are also conserved (Li et al. 2005). Among the three predicted sites on Sstp3, the yeast homologue of OstStt3, the very same potential glycosylation site has been found to be glycosylated in Drosophila as in the yeast counterpart (Table I and (Li et al. 2005)). Moreover, removal of this particular N-glycosylation site in yeast leads to lethality, indicating the essential nature of this modification for cell viability. Our results confirmed this finding, underlying the evolutionary conservation and essential function of this N-linked glycan, which also predicts the importance of this modification for all other eukaryotic oligosaccharyltransferase 3 homologues.
Another glycoprotein, for which the importance of N-glycosylation has already been implicated, is rhodopsin (also referred to as neither inactivation nor afterpotential E, ninaE) an evolutionary conserved G-protein coupled receptor protein with two potential glycosylation sites, Asn-20 and Asn-196. Interestingly, previous studies indicated that Drosophila rhodopsin undergoes a temporal N-glycosylation which is present only on newly synthesized protein, but not on the mature one (de Couet and Tanimura 1987; Huber et al. 1990). The importance of N-glycosylation for rhodopsin maturation was also supported by in vivo experiments using modified rhodopsin with one of the potential glycosylation sites (Asn-20) removed (O’Tousa 1992). These previous experiments found that N20I substitution resulted in low rhodopsin levels and age-dependent degeneration of the photoreceptors (O’Tousa 1992). All these data suggest the importance of N-linked glycosylation for rhodopsin functioning. Here, we experimentally confirmed the utilization of the second N-glycosylation site, Asn-196 (Table I), which is located in the extracellular loop between the 4th and 5th transmembrane domains of Drosophila rhodopsin. This glycosylation site may be highly relevant to the biological function of this molecule, which can now be tested experimentally using in vivo approaches.
Our study identified N-glycans on a number of glycoproteins that are known to participate in larger protein complexes (Table I). Examples include the septate junction complex components neurexin IV, neuroglian, contactin, and gliotactin (Genova and Fehon 2003; Faivre-Sarrailh et al. 2004). Likewise, we found N-glycosylated sites on basigin and integrins that are also known to colocalize in the eye tissue (Curtin et al. 2005). In the light of these findings, it is interesting to mention an earlier hypothesis about the possible role of lectin-type interaction between heterophilic cell adhesion molecules that involve oligomannosidic glycans on these molecules (e.g., interaction between L1 and N-CAM molecules, where the 4th IgG domain of N-CAM harbors a lectin-like domain that interacts with the oligomannosidic glycans of L1) (Horstkorte et al. 1993). Likewise, mouse basigin was also found to contain a carbohydrate recognition domain (CRD) and to bind brain glycoproteins that are known to carry oligomannosidic glycans (Heller et al. 2003). Whether this principle of carbohydrate-assisted recognition/binding would apply to other cell adhesion molecules remains to be determined, although the relatively high proportion of oligomannosidic glycans (Gurd and Fu 1982) and mannose binding lectin proteins in the CNS (Zanetta et al. 1978) would certainly be supportive of this notion.
Yet another group of glycoproteins identified in the present study are the glutamate channel proteins. Interestingly, the lectin Con A has long been used in electrophysiological recordings in both vertebrate and invertebrate preparations for the suppression of ionotropic glutamate channel desensitization (Standley and Baudry 2000). The N-glycans present on these glutamate receptor proteins are not required for the basic function of these channels per se, as the recombinant receptors without N-glycans are still able to function as ion channels (Everts et al. 1997). However, the N-glycans that are present on the extracellular domains are required for Con A mediated regulation. Having mapped these sites will now allow the use of advanced genetic tools to address their in vivo function in Drosophila. These results will likely be important for understanding the role of glycosylation of glutamate channels in other organisms, including mammals, since oligomannosidic structures predominate on the NMDA and AMPA receptor subunits in the brains of a broad range of species examined so far (Clark et al. 1998).
Our results contain information about several novel proteins with unknown functions which are currently not assigned to any biochemical pathway and subcellular compartment. Identifying these proteins as N-glycosylated allows us to conclude that they are targeted to the secretory pathway. For example, no specific molecular functions or subcellular localization are currently known for the protein products of vir-1, halfway, iris or yellow-c (among numerous others, see Table I). Our results now suggest that these proteins are membrane-associated or secreted products, residing in or modified through the secretory pathway.
Analysis of the amino acids surrounding the consensus N-glycosylation site revealed that the N-X-T site is utilized 67% of the time, while the N-X-S site 33% of the time in our glycopeptide sample. This finding is in agreement with the result of previous analyses of N-linked consensus sequence that used information from databases of metazoan protein sequences (Ben-Dor et al. 2004; Petrescu et al. 2004). When adjusted to the frequencies of the corresponding S and T codons in the Drosophila genome (see Materials and methods), the bias toward glycosylating N-X-T as opposed to N-X-S becomes even more apparent, suggesting that the probability of utilization for N-X-T sequon is more than three times higher than that for N-X-S. We also detected a strong preference for Thr downstream of the sequon (position +4), while Ser was significantly disfavored there (position +3) (Figure 6A), which may reflect a better "conformational fitness" of Thr over Ser for N-glycosylation machinery in the vicinity of glycosylation site.
In agreement with previous studies, we also found a marked preference for hydrophobic amino acids in the closest proximity upstream and downstream of the glycosylation site, and in particular, a preference for aromatic amino acids at the two positions immediately preceding the site (Figure 6B and (Petrescu et al. 2004)). In addition, our results confirmed a previously detected bias in the distribution of basic amino acids around the glycosylation site, with strong preference at the upstream position –3 and significantly reduced frequency at the +3 downstream position (Figure 6B and (Petrescu et al. 2004)). The strongest biases found in our study were clustered around the N-X-T/S sequon, with the exception for aromatic residues (Figure 6B), the occurrence of which was also found to be positively biased at relatively distant sequence positions (–18, –16, +16, +19). These distant positions may, however, be close in space to the glycosylation site due to protein conformation (Petrescu et al. 2004). Overall, our data are consistent with the hypothesis that N-linked glycosylation occurs frequently at sites with low accessibility in the folded protein (high incidence of hydrophobic residues), while mostly local interactions in the close proximity of the glycosylation site (strong biases are clustered around the sequon) influence the glycosylation process (Petrescu et al. 2004). In addition, a significant preference for the presence of aromatic residues along the entire range of amino acids analyzed (–20 to +20) supports the idea that glycosylation promotes folding by affecting more distant aromatic residues, thus serving as a nucleation site and stabilizing protein conformation (Jitsuhara et al. 2002; Petrescu et al. 2004).
Interestingly, we found neither a strong inhibitory effect of proline in position immediately downstream of the N-X-T/S consensus (Gavel and von Heijne 1990; Ben-Dor et al. 2004; Petrescu et al. 2004) nor a notable reduction in the frequency of acidic residues immediately before the glycosylation site (Petrescu et al. 2004), both of which were noted in the previous studies. These differences may reflect specific properties of the insect N-glycosylation mechanism, since very few insect glycoproteins were included in the previous analyses. Another explanation might be that our sample size did not allow us to decipher some biases at a statistically significant level. Nevertheless, our study adds a significant amount of new sequence information to the collection of experimentally verified nonredundant N-glycosylation sites used in previous analyses (Ben-Dor et al. 2004; Petrescu et al. 2004).
It is worth emphasizing that although the large number of identified glycoproteins (205) from Drosophila heads is unprecedented, this is not an exhaustive database of N-glycosylated glycoproteins expressed in Drosophila brain. Glycopeptides that in addition to the N-glycans also carried O-glycans or other modifications might be missed by this method due to the high complexity of mass spectrometry (MS) sequencing profiles for such peptides. Also, low-abundant glycopeptides might have fallen below the detection threshold of the mass spectrometer or generated MS/MS fragmentation patterns of insufficient robustness, and hence could not be identified. Our stringent filtering to achieve 99% confidence at the peptide level, while reducing our false-positives (only 2 out of 309 N-linked sites assigned were not in the established N-X-S/T sequon and thus were discarded as false-positives), almost certainly resulted in the discarding of valid glycopeptides. In addition, although Con A and WGA are perhaps among the most suitable lectins to recover the largest proportion of N-glycosylated peptides, there are likely some peptides with N-glycan structures that do not bind efficiently to any of the lectins utilized in our study. Finally, the use of PNGaseF to release glycans and tag glycosylation sites precluded the identification of peptides that carry HRP-epitopes, which have been shown to account for slightly less than 1% of the total glycan profile in whole Drosophila embryos (Aoki et al. 2007).
Given that GlcNAcT-II and DSiaT, glycosyltransferases involved in the biosynthesis of hybrid/complex type structures are almost exclusively expressed in the nervous system of Drosophila (Tsitilou and Grammenoudi 2003; Koles et al. 2004), it is somewhat surprising that only a relatively small fraction of N-glycans identified have more extensively processed termini (Figure 3 and 4), similar to the result from analysis of total glycans from the embryonic stage (North et al. 2006; Aoki et al. 2007). This result may indicate that the role of these structures in the adult CNS is highly specialized and, therefore, restricted to a limited number of glycoproteins.
The sialylated glycan structure that we detected in this study is identical to the previously reported sialylated glycan from embryos (Aoki et al. 2007). Again, we did not find any evidence for the presence of LacdiNAc (GalNAcß1,4GlcNAc) or sialylated LacdiNAc termini, despite the fact that in in vitro assays, two Drosophila ß4Gal transferase homologues, ß4GalNAcTA and ß4GalNAcTB, transfer GalNAc as opposed to Gal residues, while DSIAT enzyme shows notable preference for LacdiNAc over LacNAc termini (Koles et al. 2004; Haines and Irvine 2005). Thus, our investigation of N-linked glycans and the previous study of embryonic glycans, both indicate that the most prevalent complex termini of N-linked chains are LacNAc or sialylated LacNAc structures, while LacdiNAc and sialyl-LacdiNAc, if present at all, represent a minute fraction of highly processed N-glycans. These results also suggest that ß4GalNAcTA and/or ß4GalNAcTB enzymes in vivo may have mainly ß4Gal-transferase activity toward N-glycans. Another possibility arises from recent studies suggesting that both ß4GalNAcTA and ß4GalNAcTB are predominantly involved in glycolipid biosynthesis (Chen et al. 2007), and thus another, yet unidentified enzyme may be responsible for the synthesis of LacNAc termini on N-linked glycans. Whether LacdiNAc structure is synthesized on Drosophila glycoproteins in vivo, and what would be the potential function of this structure, still remains to be determined.
In conclusion, the data presented in this paper identify a large repertoire of N-glycosylation sites that are occupied in vivo, as opposed to using in silico predictions that may not necessarily reflect the in vivo situation. Therefore, these data directly enable future functional studies of a large number of N-glycosylation sites in the nervous system of Drosophila melanogaster, which will lead to a more thorough understanding of the role of this important posttranslational protein modification.
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Materials and methods |
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Materials
Agarose conjugated lectins Con A, WFA, WGA, and SNA were from Vector Laboratories (Burlingame, CA). L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated bovine pancreas trypsin was from Sigma (St. Louis, MO). PNGaseF was purchased from Prozyme, San Leandro, CA. SepPak C18 cartridges were from Waters (Milford, MA). Wild-type Drosophila melanogaster Oregon-R strain is available from the Bloomington Stock Center (Indiana University) and various Drosophila research laboratories.
Fly head collection and membrane fractionation
Wild-type Oregon-R flies were amplified in large, one-gallon containers, which were prepared from recycled heat-sterilized milk-jugs that had air-windows cut in them (design details are available upon request). These were maintained at room temperature on a 12:12 hour light:dark cycle, and adult flies were collected and stored at –80°C. Heads (50 g final) were prepared by vortexing the frozen flies (to separate the heads from the bodies) and this mixture was applied to a precooled assembly of four sieves (from the top: 800, 600, 355, and 106 µm) (Matthies and Broadie 2003). Adult bodies were retained by the top two sieves and heads were collected from the 3rd sieve, and immediately pulverized in liquid nitrogen. The sample was then further homogenized in a glass homogenizer in 9 w/v 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4 containing 1 mM CaCl2, MgCl2 and 1 mM phenylmethanesulphonylfluoride (PMSF). The homogenate was first centrifuged at 1500 x g for 10 min at 4°C to remove nuclei and cuticle debris. The resulting supernatant was centrifuged at high speed (100,000 x g) for 2 h at 4°C and the resulting pellet (representing most of the membranous material) was used for the isolation of glycopeptides. The supernatant from this centrifugation step (representing the fraction of soluble molecules not associated with the membranes) was not further analyzed in this study.
Glycopeptide preparation
The membrane pellet was rinsed and then resuspended in 50 mM ammonium bicarbonate buffer and the solution was reduced in 10 mM dithiothreitol (DTT) for 2 h at room temperature (some precipitation occurred). Iodoacetamide to a final concentration of 20 mM was then added and incubated for another 2 h at RT in the dark. Finally, the solution was dialyzed extensively against 10 mM NH4HCO3. Lipids were removed by extracting twice with chloroform:methanol:dialysate (4:8:3) while stirring overnight at 4°C. The lipid-free protein pellet was dried extensively under a gentle stream of nitrogen. The dried pellet was sonicated in 20 mM NH4HCO3 and digested with trypsin for 48 h at 37°C (two additions of trypsin at 2.5% w/w). The digest was then cleared by centrifugation and the supernatant was incubated at 100°C for 10 min to inactivate trypsin and then filtered through a 100 kDa cutoff filter/concentrator to remove all molecular components larger than 100 kDa.
Lectin affinity chromatography
The tryptic digest was adjusted with lectin buffer to give 10 mM Tris pH 7.4, 150 mM NaCl and 1 mM CaCl2 and MgCl2. The peptides were then fractionated according to standard protocols using Con A, WFA, SNA, and WGA agarose columns. Before the elution step, the columns were washed with five column volumes of lectin buffer. The following elution conditions were used: ConA weakly bound fraction was eluted with 10 mM methylglucoside; Con A strongly bound fraction – with 100 mM -methylmannoside that was preheated to 60°C (Sherblom and Smagula 1993); WFA fraction – with 100 mM GalNAc in lectin buffer: SNA fraction – with 20 mM unbuffered ethylenediamine; and WGA fraction – with 0.2 M GlcNAc in 10 mM Tris pH 7.4. The fractions eluted from lectin columns were desalted on an analytical Superdex Peptide column (Amersham, Piscataway, NJ) using Akta Pharmacia FPLC and stored frozen prior to subsequent analyses.
N-linked glycan profiles
Portions of the affinity-purified glycopeptide fractions were dried and resuspended in 50 µL of 20 mM sodium phosphate buffer, pH 7.5, for digestion with PNGaseF. Following PNGaseF digestion for 18 h at 37°C, released oligosaccharides were separated from peptide and enzyme by passage through a Sep-Pak C18 cartridge. The digestion mixture was adjusted to 5% acetic acid and loaded onto the Sep-Pak. The column run-through and an additional wash with three column volumes of 5% acetic acid, containing released oligosaccharides were collected together and evaporated to dryness. To facilitate analysis by MS, released oligosaccharide mixtures were permethylated according to the method of Ciucanu and Kerek (Ciucanu and Kerek 1984).
For mass analysis by NSI-MSn, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer (LTQ, Thermo Finnigan) using a nanoelectrospray source at a syringe flow rate 0.40– 0.60 µL/min. The capillary temperature was set to 210°C and MS analysis was performed in positive ion mode. For fragmentation by CID in MS/MS and MSn modes, 28% collision energy was applied. As previously described, the TIM functionality of the Xcalibur software package (version 2.0) was utilized to detect and quantify the prevalence of individual glycans in the total glycan profile (North et al. 2006; Aoki et al. 2007). All of the MS/MS spectra produced from each TIM analysis were manually examined for the presence of fragment ions indicative of glycan. Most permethylated oligosaccharides were identified both as singly and as doubly-charged species by NSI-MS. Peaks in TIM or full MS scans were quantified if threefold or greater above background. Glycan prevalence was calculated as "% Total Profile" where the total profile was taken as the sum of the peak intensities for all quantified glycans.
Peptide analysis
A portion of individual lectin-bound glycopeptide fractions was digested with PNGaseF in H218O to introduce a mass shift of 3 daltons at the previous site of N-linked glycosylation (+1 Da shift for Asn to Asp conversion, +2 Da shift for the incorporation of one 18O into the resulting Asp side chain, (Kuster and Mann 1999; Kaji et al. 2003). Following evaporation to dryness, peptides were resuspended in 40 mM NH4HCO3 and incubated with 1 µg trypsin at 37°C for 4 h to allow any 18O that may have added to the C-terminus of the tryptic peptides to be exchanged for 16O (Angel et al. 2007). Peptides were again evaporated to dryness and stored at –20°C until analyzed. The peptides were resuspended with 20 µL of mobile phase A (0.1% formic acid, FA, in water) and filtered with 0.2 µm filters (Nanosep, PALL, East Hills, NY). The samples were loaded off-line onto a nanospray tapered capillary column/emitter (360 x 75 x 15 µm, PicoFrit, New Objective, Woburn, MA) self-packed with C18 reverse-phase (RP) resin (8.5 cm, Waters) in a Nitrogen pressure bomb for 10 min at 1000 psi (
5 µl load) and then separated via 160-min linear gradient of increasing mobile phase B (80% acetonitrile, ACN, and 0.1% formic acid in water) at a flow rate of 200 nL/min directly into the mass spectrometer. One-dimensional (RP separation) LC-MS/MS analysis was performed on a Finnigan LTQ mass spectrometer (Thermo Electron Co., San Jose, CA) equipped with a nanoelectrospray ion source. A full MS spectrum was collected (m/z 350–2000) followed by eight MS/MS spectra following CID (34% normalized collision energy) of the most intense peaks. Dynamic exclusion was set at 2 for 30 s exclusion. The resulting data was analyzed using Sequest requiring fully tryptic peptides and allowing for dynamic modification of Cys +57 (alkylation with iodoacetamide), Met +16 (oxidation), and Asn +3 (site of N-linked glycosylation) against both a forward and reverse nonredundant D. melanogaster database and stringent filtering applied to generate a false-discovery rate of 1% at the peptide level. The consensus sequence (N-X-S/T) was not used to initially filter the data, and 307 of 309 assigned modified Asn were in this established sequon for N-linked glycosylation, further demonstrating the reliability of this approach to map N-linked glycosylation sites. The two sites assigned that were not in the consensus sequence were discarded as likely false-positives. Spectra for any of the reported peptides are available upon request.
Analysis of amino acid biases around N-glycosylation site
The sequences of 307 peptides with experimentally confirmed N-glycosylation were aligned according to their glycosylation site. Occurrence of each amino acid at every position within the range of [–20; +20] relative to the asparagine (Asn) of the glycosylation site was calculated and compared to the theoretical occurrence expected in a random peptide sequence. The random occurrence was estimated based on the frequencies of corresponding codons in the Drosophila genome (Nakamura et al. 2000), and assuming that amino acids are distributed at each position according to a binomial distribution (the probability to find a certain amino acid residue at any position in a random peptide sequence was determined by the sum of frequencies of corresponding codons). The mean, variance, and standard deviation of binomial distribution for each amino acid was calculated, and the theoretical occurrence was compared to the experimental one at each position within the [–20; +20] range of residues around the glycosylation site. A difference in experimental occurrence from the random occurrence was considered a weak bias if it was greater than 2 SD but less than 3 SD; and it was considered a strong bias if the difference was greater than 3 SD. To facilitate a more statistically significant conclusion, some amino acids with similar physicochemical properties were analyzed in small groups as follows: (D, E), (I, L, V), (K, R). In addition, the analysis was also performed for several major types of amino acids, according to their physicochemical properties: G, A, V, L, I, M – hydrophobic; S, T, N, Q, C – polar, uncharged; K, R, H – basic; D, E – acidic; W, Y, F – aromatic (Petrescu et al. 2004; Nelson and Cox 2005). It is worth pointing out that according to the theorem of De Moivre-Laplace (Korn and Korn 1968), the binomial distributions for the grouped or individual amino acids could be well approximated by normal distributions (with the exception for individually analyzed C, H, M, W, and Y), and thus, the weak and strong biases approximately correspond to the 95% and 99% confidence levels, respectively.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) grant (GM069952 to V.M.P., GM072839 to M.T.); scientist development grant (0535220N to L.W.) from the American Heart Association.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We acknowledge the help of Stacey Withman, Theodore Markulin, and Shawn Hanrahan with the Drosophila populations and fly collection. We thank Dr. Larry Dangott for his expert assistance with peptide chromatography, and everyone in the Department of Biochemistry & Biophysics at Texas A&M University who generously contributed to our jug collection as well as Coffee Station and StarbucksTM coffee shops in College Station for donating their milk-jugs for this project. We also thank reviewers of the manuscript for their helpful comments. L.W. is a Georgia Cancer Coalition Distinguished Scientist
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Abbreviations |
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CNS, central nervous system; Con A, Concanavalin A; DTT, dithiothreitol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LacdiNAc, GalNAcß1,4GlcNAc; LacNAc, Galß1,4GlcNAc; MS, mass spectrometry; PMSF, phenylmethanesulphonylfluoride; PNGaseF, N-glycanase; SD, standard deviation; SNA, Sambucus nigra lectin; TIM, total ion mapping.; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; WFA, Wisteria floribunda lectin; WGA, wheat germ agglutinin
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