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. 2014 Nov 10;25(4):403–411. doi: 10.1093/glycob/cwu122

Conserved ion and amino acid transporters identified as phosphorylcholine-modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans cells

Casey J Snodgrass 1, Amanda R Burnham-Marusich 1, John C Meteer 1, Patricia M Berninsone 1,1
PMCID: PMC4339876  PMID: 25387872

Abstract

Phosphorylcholine (PC) modification of proteins by pathogens has been implicated in mediating host–pathogen interactions. Parasitic nematodes synthesize PC-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections. Only two nematode PC-modified proteins (PC-proteins) have been unequivocally identified, yet discovering the protein targets of PC modification will be paramount to understanding the role(s) that this epitope plays in nematode biology. A major hurdle in the field has been the lack of techniques for selective purification of PC-proteins. The nonparasitic nematode Caenorhabditis elegans expresses PC-modified N-linked glycans, offering an attractive model to study the biology of PC-modification. We developed a robust method to identify PC-proteins by metabolic labeling of primary embryonic C. elegans cells with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification and subsequent high-throughput LC–MS identification of propargyl-labeled proteins. All proteins identified using stringent criteria are known or predicted to be membrane or secreted proteins, consistent with the model of a Golgi-resident, putative PC-transferase. Of the 55 PC-N-glycosylation sites reported, 33 have been previously observed as N-glycosylation sites in high-throughput screens of C. elegans. Several identified PC-proteins are nematode-specific proteins, but 10 of the PC-proteins are widely conserved ion transporters and amino acid transporters, while eight are conserved proteins involved in synaptic function. This finding suggests a functional role for PC-modification beyond immunomodulation. The approach presented in this study provides a method to identify PC-proteins in C. elegans and related nematodes.

Keywords: Caenorhabditis elegans, click chemistry, N-glycosylation, phosphorylcholine

Introduction

Phosphorylcholine (PC) modifications of proteins or lipids by prokaryotic and eukaryotic pathogens have been implicated in facilitating the pathogens' evasion of the host immune system (Grabitzki and Lochnit 2009) and in altering host–cell function (Mukherjee et al. 2011; Tan et al. 2011). Parasitic nematodes synthesize PC-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections (Grabitzki and Lochnit 2009). Only two nematode PC-modified proteins (PC-proteins) have been unequivocally identified, yet discovering the protein targets of PC modification will be paramount to understanding the role(s) that this epitope plays in nematode biology. ES-62, the best-characterized PC-protein, is an excreted–secreted product of the rodent parasite Acanthocheilonema viteae (Houston and Harnett 2004) and has an immunomodulatory role that is partially dependent on its modification with PC (Harnett et al. 2008). Interestingly, the incidence of an auto-immune and an inflammatory disease, type I diabetes and multiple sclerosis, respectively, are reduced in areas of endemic nematode infection (reviewed in Zaccone et al. 2006). Thus, PC-proteins such as ES-62 are being investigated for their therapeutic potential in treating inflammatory and auto-immune disorders (Harnett and Harnett 2006).

The free-living nematode Caenorhabditis elegans synthesizes PC-modified N-glycans (reviewed in Haslam and Dell 2003) and glycolipids (Gerdt et al. 1999) and thus offers an attractive model to investigate the biology of PC-modified molecules. Several studies have demonstrated the attachment of PC to N-glycans in nematodes and elucidated their structures (Cipollo et al. 2002, 2005; Paschinger et al. 2004; Hanneman et al. 2006). To date though, aspartyl protease 6 (ASP-6) remains the only C. elegans protein with substantial evidence for its PC modification (Lochnit et al. 2006). Furthermore, PC modification in nematodes occurs when a putative transferase resident to the Golgi utilizes phosphatidylcholine as a PC donor to decorate terminal N-acetylglucosamine (GlcNAc) residues of N-linked glycoproteins (Cipollo et al. 2004; Houston and Harnett 2004). Protein N-glycosylation occurs in the secretory pathway, but surprisingly few membrane or secreted PC-proteins were identified in a proteomics screen in C. elegans, which instead identified several putative cytosolic, nuclear and mitochondrial PC-proteins (Grabitzki et al. 2008). The fact that PC-modified N-glycans have been detected in C. elegans, yet the proteins to which they are attached remain largely unidentified, highlights the need for more robust methods to identify PC-proteins, especially those with low expression.

Results

We sought to develop a chemical biology approach to detect, purify and identify C. elegans PC-proteins with high specificity. The impermeability of the nematode cuticle results in the relative inaccessibility of C. elegans somatic cells to metabolic labeling in vivo. In fact, the incorporation of azido-labeled sugars and detection reagents in intact C. elegans nematodes is restricted to certain cells and tissues (Laughlin and Bertozzi 2009). The low incorporation efficiency of azido-precursors in vivo is therefore not sufficient for downstream glycoprotein identification. We chose C. elegans primary cell cultures as a proof-of-principle model because they are amenable to metabolic labeling with azido-tagged sugar analogs, enabling Click Chemistry detection and mass spectrometry identification (Burnham-Marusich et al. 2012). They can also be isolated in quantities large enough for biochemical analysis (Burnham-Marusich et al. 2012), thereby offering a viable model to circumvent the drawbacks of in vivo metabolic labeling. We aimed to extend the use of these primary cell cultures for the study of PC-proteins, by labeling PC-proteins with propargylcholine (PG-choline). PG-choline is an alkyne-modified choline analog (Figure 1A), which was recently shown to incorporate into phosphatidylcholine in NIH 3T3 cells (Jao et al. 2009). The pathway by which choline is converted to phosphatidylcholine is conserved between mammals and C. elegans (Lochnit and Geyer 2003; Gibellini and Smith 2010) (see Kennedy pathway, Figure 1B). Thus, we reasoned that if C. elegans primary embryonic cells metabolize PG-choline into PG-phosphatidylcholine via the conserved Kennedy pathway (Houston et al. 2008), then the putative PC-transferase could utilize the resulting PG-phosphatidylcholine as a donor for PC-modification of N-glycoproteins (Figure 1B).

Fig. 1.

Fig. 1.

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Phosphatidylcholine biosynthesis in C. elegans. (A) The structures of choline and PG-choline are shown. (B) The de novo choline or Kennedy pathway (gray box) converts choline (Cho) into phosphatidylcholine (PtdCho). The Kennedy enzymes are found ubiquitously in eukaryotes, except in Giardia lamblia. The nematode phosphobase methylation pathway (white box) converts phosphoethanolamine (pEA) into phosphocholine (PCho). The multiple methylation of pEA to PCho also occurs in plants and Plasmodium falciparum, catalyzed by a single phosphoethanolamine methyltransferase, PEAMT.

Therefore, C. elegans primary embryonic cells were isolated and labeled with 100 or 500 µM PG-choline (+) or choline (−) as a negative control. After 72 h, cells were harvested and the protein fraction extracted. The alkyne moiety of PG-choline allows for highly selective conjugation with biotin-azide via Click Chemistry (Figure 2A). PG-choline-labeled proteins reacted with biotin-azide were readily detected by avidin::HRP western blot (Figure 2B) in stark contrast to the control. The avidin::HRP signal at a diversity of molecular weights recapitulated the western blot pattern observed using the PC reactive antibody TEPC-15 (Paschinger et al. 2006; Grabitzki et al. 2008), indicating that PG-choline was metabolically incorporated into multiple proteins by C. elegans embryonic cells. Although some PC-modified glycoproteins are expected to be secreted, our attempts to isolate and identify propargylcholine-modified proteins from the spent medium after incubation of the cells were not successful. This is due to their very low abundance relative to the fetal bovine serum (FBS) proteins present in the medium as the cells are cultured in media containing 10% FBS. However, all secreted and excreted proteins are posttranslationally modified as they traffic through the endoplasmic reticulum and Golgi apparatus. Therefore, PC-proteins isolated from cell lysates are expected to include secreted/excreted proteins as they traffic through the secretory pathway.

Fig. 2.

Fig. 2.

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Propargylcholine is metabolically incorporated into C. elegans N-glycoproteins. (A) Schematic showing a propargylcholine (PG-choline)-labeled N-glycoprotein (1) and its conjugation with biotin-azide (2) via Cu(I)-catalyzed Click Chemistry to form a biotin-conjugated, PG-choline-labeled protein (3). (B) Proteins from C. elegans embryonic cells metabolically labeled with PG-choline (+) or choline (−) were reacted with biotin-azide and analyzed by western blot with avidin::HRP. (C) PG-choline-labeled C. elegans cell lysate was reacted with biotin-azide, digested with PNGase F (+) or mock digested (−), then analyzed by western blot with avidin::HRP. (D) COS-7 cells were metabolically labeled with PG-choline (+), choline (−), azido-GalNAc (GalNAz, +) or GalNAc (−). Cell lysates were reacted with biotin-azide or biotin-alkyne and analyzed by western blot with avidin::HRP. (B–D, right panels) Sypro Ruby was used to confirm protein loading. The PC-N-glycan structure shown was modified from Cipollo et al. (2005).

Since PC is known to elaborate N-glycans, we next tested if PG-choline specifically labeled PC-modified N-glycoproteins by enzymatically removing N-glycans with PNGase F. Caenorhabditis elegans proteins metabolically labeled with 250 µM PG-choline were reacted with biotin-azide and either treated with PNGase F (+) or mock treated (−). Probing with avidin::HRP revealed that PNGase F digestion substantially decreased the biotin label, indicating that a majority of PG-choline was attached to proteins via PNGase F-sensitive N-glycans (Figure 2C).

Both C. elegans and mammals convert choline into phosphatidylcholine via the conserved CDP-choline branch of the Kennedy pathway (Houston et al. 2008; Gibellini and Smith 2010) (Figure 1B). In mammalian cell cultures, propargylcholine is efficiently incorporated into all classes of choline phospholipids and replaces >50% of the choline head groups in total lipids by 24 h (Jao et al. 2009). However, unlike C. elegans, mammalian cells do not modify N-glycoproteins with PC and therefore are not expected to incorporate propargylcholine into proteins. COS-7 cells were incubated with 250 µM PG-choline (+) or choline (−), reacted with biotin-azide, and detected with avidin::HRP. Biotin signal was undetectable in both COS-7 PG-choline- and choline-labeled protein samples (Figure 2D). As a positive control, COS-7 cells were metabolically labeled in parallel with N-acetylgalactosamine (GalNAc) or peracetylated azido-GalNAc (GalNAz) and reacted with biotin-alkyne. GalNAz labeling resulted in robust avidin::HRP detection of biotin-tagged metabolically labeled proteins when compared with undetectable avidin::HRP signal in the GalNAc control, which indicates successful incorporation of the sugar into glycoproteins and that the cells were metabolically active (Figure 2D). Labeling of two additional mammalian cell lines, HeLa and JEG-3, also resulted in undetectable PG-choline incorporation into proteins (Supplementary data, Figure S1A and B). Since the signal was only detected in the C. elegans samples, these data together with the PNGase F experiment suggest that the PG-choline incorporation into C. elegans proteins is specific to the elaboration of N-linked moieties of PC-labeled glycoproteins.

Next, the feasibility of purifying PG-choline-labeled C. elegans proteins was assessed by applying, in parallel, PG-choline (+)- and choline (−)-labeled cell lysates reacted with biotin-azide to streptavidin beads. Aliquots of the starting material (input), unbound proteins and eluted proteins were analyzed by avidin::HRP Western blot. As before, robust biotin labeling was detected in the PG-choline input sample but not in the choline control (Figure 3A). A clear reduction of biotin detection in the unbound protein sample indicated efficient capture of PG-choline-labeled proteins. The biotin signal of the PG-choline elution lane was approximately equal to that of the input sample despite undetectable protein staining (Figure 3B), demonstrating enrichment of PG-choline-labeled proteins in the elution fraction.

Fig. 3.

Fig. 3.

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Propargylcholine-labeled proteins can be purified and identified by mass spectrometry. (A) Caenorhabditis elegans PG-choline (+)- or choline (−)-labeled cell lysates were reacted with biotin-azide and applied to streptavidin beads. Three percent of the input sample, 3% of the unbound sample and 10% of the eluted sample were analyzed by western blot with avidin::HRP. (B) Sypro Ruby was used to confirm sample loading. (C) PG-choline-labeled proteins reacted with biotin-azide were captured onto streptavidin beads. (D) Trypsin digestion released non-PG-choline-modified, tryptic peptides from the PG-choline-labeled peptides. (E) PNGase F digestion released the bead-bound PG-choline-labeled peptides. The resulting peptide samples were subjected to LC–MS-MS analysis for protein identification and PC-N-glycosylation site identification (the latter by using peptides released by PNGase F). The PC-N-glycan structure shown was modified from Cipollo et al. (2005).

To identify C. elegans PG-choline-labeled proteins, we performed a large-scale streptavidin bead purification. After extensive and stringent washing, captured proteins were trypsin digested directly on the beads to release non-PG-choline-modified peptides from PG-choline-modified peptides (Figure 3C and D). After trypsin digestion, PNGase F was used to specifically release the PG-choline-N-glycopeptides that remained attached to the beads (Figure 3D and E). Peptides resulting from trypsin digestion and peptides from PNGase F digestion were analyzed by LC–MS-MS. PNGase F cleaves between the innermost GlcNAc and Asn residues of high mannose, hybrid and complex oligosaccharides of N-linked glycoproteins, deamidating the modified Asn to Asp and resulting in a +0.98 Da mass shift at that residue (Zhao et al. 2010). Thus, potential sites of PG-choline-N-glycan attachment were identified by including this mass shift as a variable modification in the MS analysis and verifying that the deamidated Asn occurred at an N-glycosylation consensus sequence, N-X-[S/T], where X≠P (Table I; Supplementary data, Dataset 2).While the Asn to Asp signature after PNGase F treatment has been widely used to identify N-glycosylation sites, recent studies have shown that in vivo deamidation and chemical deamidation during sample preparation can contribute to false-positive N-glycosylation site assignment, with the majority of the deamidation events occurring where the deamidated Asp is followed by amino acids with small and hydrophilic side chains (Gly and Ser) (Palmisano et al. 2012). To assess the rate of nonenzymatic Asn deamidation during our sample preparation, we analyzed the peptides released by trypsin digestion (not treated by PNGase F, and therefore not occupied by a glycan) for deamidation as a variable modification as recently described (Hao et al. 2011; Palmisano et al. 2012). The Asn deamidation rate in the tryptic digest (number of unique Asn deamidated peptides/number of unique total peptides) was 9.7% (69/710), in the range of the 4–9% recently reported (Hao et al. 2011; Song et al. 2013). To assess the value of the enrichment method, we used the identified Asn-deamidated nonglycopeptides as a database of false positives to remove from the dataset of Asn-deamidated glycopeptides (i.e., Asn-deamidated peptides identified after PNGase F treatment). Comparison of these two datasets revealed no Asn deamidated peptides in common. This supports the stringency of the streptavidin enrichment step in our protocol, which significantly reduces the false discovery rate.

Table I.

Caenorhabditis elegans phosphorylcholine-modified proteins and their modification sites identified by propargylcholine labeling

Protein name Accession number Pc-N-glycosylated peptide(s) Tryptic peptides Tryptic peptide coverage (%) Function/expression
Adenylyl cyclase (acy-1) gi|17551720 (K)HLVEEQDTCN933VTAIMIPPIRK(G) 3 5.3 Neurons and muscle cellsd
Amino acid transporter (atgp-1) gi|32565753 (R)YVQAVN535NTATGSTFIVALNFGDKEQK(I)
(K)DLLNAEISVVTAN584VTDYR(V)a,b 		15 47.0 Amino acid transporter subunit (Veljkovic et al. 2004)
Amino acid transporter (atgp-2) gi|25147709 (R)FHVDSYSN351ESTIADGKK(I)a 8 25.0 Amino acid transporter subunit (Veljkovic et al. 2004)
Anion/bicarbonate transporter (abts-1) gi|17507499 (K)STGLPIAFN615DTFIDYTAAN625LTDCR(T)a,b/a,b 12 17.0 Ion transporter; neurons and muscle cells (Bellemer et al. 2011)
Dystroglycan (dgn-1) gi|17551334 (R)VFIGELFEHNLGDN35YTIATGK(D)a,b,c
(R)IYSFN142FSLINR(E)c,b 		4 13.0 Basement membrane component; epithelia and neurons (Johnson et al. 2006)
Na/K ATPase-β (nkb-1) gi|17505629 (K)TYLTKYDSN140ATETR(E)a,b,c 15 56.0 Transporter
Neprilysin-2 (nep-2) gi|32563993 (K)FN284FTSLLVNSR(R) 2 5.4 Neurons and muscle cellsd
Hypothetical protein (T22B11.4) gi|71994330 (R)TAGGCTCAHTLAANCHTDLAQTQHPAN108LSMK(T)a
(R)HLKIEN157VTLHYK(F)a,b 		5 15.0 Expressed in pharynx, pharyngeal glandsd
Hypothetical protein (Y92H12BR.3) gi|86561639 (K)LTSFIN102VTSGECIGN111VSKPDLFK(I)a,b,c/a,b,c
(K)FAN461NTLMVQIK(D)b
(K)KPGNVVGYN537YTTELTTAEIFK(L)b 		12 26.0 Unknownd
K/Cl cotransporter (kcc-2) gi|193206392 (K)TQVQPN328CTADGLQDLFCSTN342GTCDHYYDR(M) 4 2.1 Ion transporter; neurons and muscle cells (Bellemer et al. 2011)
Lysosome-associated membrane protein (LAMP) (lmp-1) gi|17550086 (K)FTVTFN60ETVSVEGDCNGVR(N)
(R)NN75QSVQTLNIK(F)
(K)TSNVIAFAQMN180GTVFPTDQVYEVCYLDAR(T)		 4 29.0 Germ line, intestine, coelomocyte, neuronsd
Multidrug resistance protein 1 (mrp-1) gi|71992066 (R)WSDDAKEIALSGN1008GSSSETQIR(L)b 7 16.0 Transporter
Multidrug resistance protein 2 (mrp-2) gi|17569081 (R)ELVCGDKTELLEPGWKN23R(S)b 31 31.0 Transporter
Multidrug resistance protein 5 (mrp-5) gi|17551700 (K)GHDETTTITN790GTEFLEMK(T)b 5 6.5 Broad expression, including neuronsd
β-Integrin (pat-3) gi|17554380 (K)NCVMCQQWQTGPLN672ETACDQCEFK(V)b 16 33.0 Neurons, muscle cells (Lee et al. 2001)
Patched (ptc-1) gi|193205322 (K)EAMRN188VTGDSGPELPR(E) 3 5.8 Germ line expressiond
p-Granule protein (pan-1) gi|17554292 (K)GFTIECESASIASVSENLASLN77GTELGR(L) 10 37.0 Germ line, hypodermis, pharynx, muscled
Neurotransmitter symporter (snf-11) gi|133952791 (K)MN183WTVCSAADLSVVSPVK(E) 4 8.5 GABA transporter; neuronal function (Jiang et al. 2005)
ATP-binding transporter (ced-7) gi||2133487 (K)LSVKN193ESSEEQLLTVLR(N)
(R)FEN997GTIPEEAANFEK(I)b 		13 13.0 ABC transporter (Wu and Horvitz 1998)
Vacuolar H ATPase (vha-19) gi|17543714 (K)VVN330GSATQYTK(L)a,b 4 18.0 Subunit of V-ATPase, transporter; hypodermis, excretory cell, intestine, pharynxd
Hypothetical protein (T20D3.11b) gi|222350553 (K)TAN1752TTYSGRPYLQVVGFIDR(A) 4 3.0 Unknownd
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PG-choline-labeled proteins were captured and selectively released, as described in Materials and methods. PC-N-glycosylation sites are indicated in bold with the residue number. The amino acids flanking each identified peptide are enclosed in parenthesis. a Site previously reported by Fan et al. (2005). b Site previously reported by Kaji et al. (2007). c Site previously reported by Kaji et al. (2003). d Data from Wormbase.org.

There were nine proteins identified in both the PG-choline and the choline control samples; these were considered background and were excluded. There was one protein identified in the choline control that was not identified in the PG-choline experimental sample; this was considered a false discovery. Using a minimum threshold of two unmodified, unique tryptic peptides and one PC attachment site, 21 previously unreported C. elegans proteins were identified exclusively in the PG-choline sample (Table I; Supplementary data, Dataset 2). We also identified PC-N-glycan attachment sites for 16 additional proteins for which fewer than two unmodified, unique tryptic peptides were detected (Supplementary data, Table SI). An additional 20 putative PC-proteins were identified by detection of two or more of their unmodified, unique tryptic peptides, but their sites of PC-attachment were not observed; thus, these proteins are reported as lower confidence identifications (Supplementary data, Table SII).

Discussion

We have shown that metabolic labeling with propargylcholine is a robust and sensitive method to label C. elegans N-linked glycoproteins that is amenable to identify PC-protein candidates. Of the 55 PC-N-glycosylation sites reported, 33 have been previously observed as N-glycosylation sites in high-throughput screens of C. elegans (Kaji et al. 2003, 2007; Fan et al. 2005). Furthermore, of the 20 putative PC-proteins reported where PC-N-glycosylation sites were not identified (Supplementary data, Table SII), 14 are known or are predicted to have membrane or extracellular localization.

The identification method used in this procedure relies on the widely used Asn to Asp deamidation signature after PNGase F digestion for the identification of N-glycosylation sites. Recent studies report in vivo and chemical deamidation as pitfalls of this approach that could lead to false-positive identifications, with the majority of false positives occurring at N-G and N-S sites (Palmisano et al. 2012). A dataset of experimental false positives (identified as Asn-deamidated peptides in the nonglycosylated peptide fraction) contained no peptides in common with the Asn-deamidated peptides released by PNGase F in Table I and Supplementary data, Table SI, supporting the stringency of the streptavidin enrichment and increasing the confidence of the reported N-glycopeptides.

Notably, the PC-protein candidates reported here do not overlap with the candidate PC-proteins identified by Grabitzki et al. (2008) of which 88% were cytosolic, nuclear, mitochondrial or proteins with non-positive subcellular predictions. While genuine PC modification of proteins outside of the secretory pathway cannot be ruled out, the minor percentage of secreted and membrane proteins in their candidate list may also be explained by their approach, in which C. elegans proteins were fractionated by 2D gel electrophoresis (2-DE) and identified by western blot with the PC-specific antibody TEPC-15. The poor ability of 2-DE to resolve hydrophobic membrane proteins may bias against their identification (Rabilloud, 2009). In addition, false positives cannot be ruled out due to the presence of more than one protein in a single spot. The critical advantage of the labeling approach presented in our study is that it enables affinity purification of PC-proteins from complex protein samples in the presence of harsh detergents and chaotropic agents, circumventing the shortcomings of 2-DE and enabling the identification of low abundance membrane PC-proteins. All 21 proteins identified using stringent criteria (Table I) are known or predicted to be membrane or secreted proteins, consistent with the model of a Golgi-resident, putative PC-transferase.

Among these, PC-protein identifications were the C. elegans protein, Y47A7.2, which have been previously identified as an N-linked glycoprotein (Fan et al. 2005; Kaji et al. 2007). The only detectable orthologs for Y47A7.2 are among other nematodes such as the parasitic Brugia malagi, the free-living Pristionchus pacificus, and other species from the genus Caenorhabditis (Wormbase OMA and Compara ortholog analysis). As an N-linked glycoprotein and nematode-specific protein with an ortholog in a parasitic species, Y47A7.2 represents the type of protein expected from a high-throughput identification of PC-proteins.

We note that ASP-6, the only protein previously reported as PC-modified in C. elegans (Lochnit et al. 2006), was not among the glycoproteins identified in this study. The fact that our study was performed using embryonic cells and ASP-6 expression was reported in adult worms but not earlier developmental stages (Lochnit et al. 2006) may explain this absence. The recent development of primary cell cultures from different stages of C. elegans postembryonic development (Zhang et al. 2011) should enable future studies to extend our knowledge on PC-proteins in different developmental stages.

Surprisingly, 10 of the identified PC-proteins are known or predicted transporters. These include ABTS-1, a Na+-driven Cl−−HCO3− exchanger and KCC-2, a K+–Cl− co-transporter recently reported to function in generating a cellular chloride gradient in excitable cells (Bellemer et al. 2011), the amino acid transporter subunits ATGP-1 and ATGP-2 (Veljkovic et al. 2004), the ABC transporter CED-7 involved in the engulfment of cell corpses during programed cell death (Wu and Horvitz 1998) and the multidrug resistance protein transporters MRP-1, MRP-2 and MRP-5. Moreover, eight proteins implicated in synaptic function or expressed in excitable cells were identified as PC-modified. These include dystroglycan, which is a basement membrane component expressed in epithelia and neurons (Johnson et al. 2006), the integrin-β subunit (PAT-3), which is expressed in muscle cells (Lee et al. 2001) and in neurons where it has been suggested to play a role in touch sensitivity (Calixto et al. 2010), and an Na+-dependent GABA transporter (SNF-11) expressed in neurons (Jiang et al. 2005). Additionally, PC-N-glycosylation sites within transforming growth factor β receptor (DAF-4) and netrin (UNC-6) were observed (Supplementary data, Table SI), although their modification sites are not conserved in mammals (NCBI BLASTp alignment, data not shown). Both of these proteins are expressed in neurons and have important roles regulating dauer formation (DAF-4) (Estevez et al. 1993) and axon migration (UNC-6) (Hedgecock et al. 1990).

The implications of PC modification for intracellular transport or for function of these proteins are presently unknown and will most likely have to await the identification of the PC-transferase. However, it is noteworthy that most of the PC-modified N-glycoproteins identified show a high degree of conservation along different phyla and in many nonparasitic species. This observation is consistent with a model in which the PC modification in C. elegans has a function other than immunomodulation. Evidence for a role of PC in basic cellular function is not unprecedented. Recent studies identified a PC-transferase from the intracellular pathogen Legionella pneumophila (Mukherjee et al. 2011) and showed that the activity of the host's small GTPase Rab1 is regulated by reversible addition of PC (Tan et al. 2011). In an analogous manner, PC modification of C. elegans N-glycoproteins may endow the target proteins with specific biochemical properties or altered protein–protein interactions that modulate their function. Together, the results presented here show that the N-glycoproteins substituted by PC in C. elegans comprise both widely conserved and nematode-specific proteins with intracellular as well as extracellular function. In sum, our development of a sensitive and specific method to identify PC proteins, which could also be potentially used to analyze PC-modified lipids, should enable future identification and analysis of additional PC-modified biomolecules as well as facilitate a more comprehensive understanding of the function of PC in C. elegans and related nematodes.

Materials and methods

Caenorhabditis elegans strains

N2 (wild type) and RT130 (pwIs23[vit-2::GFP]) nematodes were obtained from the CGC and maintained as described (Brenner 1974).

COS-7 cell culture and metabolic labeling

COS-7 cells were maintained in DMEM (Invitrogen) with 10% FBS (Invitrogen). The cells were metabolically labeled for 72 h with either 250 µM propargylcholine or choline, or alternatively with either 50 µM GalNAz (Invitrogen) or GalNAc (Sigma-Aldrich).

Primary embryonic cell culture

Caenorhabditis elegans primary embryonic cells were generated as described (Burnham-Marusich et al. 2012).

Cell harvesting

Cells were washed twice with PBS before adding lysis buffer [1% SDS 50 mM Tris–HCl, pH 8, with 1× protease inhibitor cocktail (CalBiochem) and 300 U/mL Benzonase (Novagen)]. Cells were lysed for 30 min on ice, and the crude cell lysate was clarified by centrifugation at 16,000 × g for 10 min.

Click chemistry detection of labeled proteins

Up to 200 µg of protein was precipitated in chloroform–methanol (1 : 4) for each reaction. The precipitated protein was resuspended in 50 µL of lysis buffer. The reaction components were then added in the order listed to a final concentration of 20 μM biotin-azide or -alkyne (Invitrogen), 50.0% DMSO, 2 mM freshly prepared ascorbic acid in water, 200 μM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (Anaspec) in DMSO and 2 mM CuSO4 in water to a final volume of 200 μL. Biotin-azide and -alkyne were used to detect propargylcholine and azido-GalNAc (GalNAz)-labeled proteins, respectively. Reactions were incubated at room temperature for 1 h, after which the samples were precipitated with chloroform–methanol (1 : 4).

Streptavidin enrichment of propargylcholine-labeled proteins

In parallel, equal amounts (1.5–3.0 mg) of PG-choline and choline-labeled C. elegans cell lysates were treated with 10 mM dithiothreitol for 15 min at 60°C and cooled to room temperature. Next, iodoacetamide was added to 20 mM and samples were incubated in the dark for 35 min at room temperature. The samples were then precipitated using chloroform–methanol (1 : 4) and reacted with biotin-azide as described in materials and methods while increasing final biotin-azide concentration to 60 µM. Samples were then precipitated using chloroform–methanol (1 : 4) and resuspended in 1.0% SDS–PBS. After the pellets were solubilized, the samples were diluted with PBS to 0.2% SDS and incubated with streptavidin-sepharose beads (GE) for 1 h with agitation. The beads were then washed as described (Nandi et al. 2006) with 0.2% SDS, 8 M urea and 1 M KCl sequentially. For analysis by western blotting, the proteins were eluted in Laemmli loading buffer with 3 mM d-biotin at 95°C for 15 min. For LC–MS-MS analysis, beads were washed three times with 50 mM NH4HCO3, pH 8.0, before adding 1.5 µg trypsin (Promega) in 50 mM NH4HCO3 and incubating 4.5 h at 37°C. The trypsin peptides were collected and the beads were washed three times with 50 mM NaPO4, pH 7.2. To elute N-glycosylated peptides attached to the beads, 500 units of PNGase F (New England Biolabs) in 50 mM NaPO4 were added and incubated for 3 h at 37°C, after which peptides were collected.

PNGase F digestion for western analysis

Up to 50 µg of protein were digested with 1250 units of PNGase F (or water for mock-treated samples) for 3 h at 37°C as per the manufacturer's recommendations (New England Biolabs).

Western blotting

Fifty micrograms of samples were resuspended in Laemmli loading buffer and analyzed by 10% SDS–PAGE unless specified otherwise. Proteins were transferred to PVDF and sample loading was assessed with Sypro Ruby protein blot stain (Invitrogen). To detect biotin-labeled proteins, membranes were incubated 1 h with 0.01–0.03 μg/mL avidin::HRP (Vector Labs) in 0.1% TBS-Tween. Membranes were washed three times for 10 min with TBS-Tween and detected by chemiluminesence.

LC–MS-MS and database searching

After reduction, alkylation and trypsin digestion, peptide samples were spiked with 100 fmol Glu-fib and separated on a Michrom Paradigm Multi-Dimensional Liquid Chromatography instrument using a Michrom Magic C18AQ 3 µ 200 Å (0.2 × 150 mm) column. Samples were analyzed on a Thermo Finnigan LTQ-Orbitrap with an Agilent ZORBAX 300SB-C18 5 µm (5 × 0.3 mm) trap. A gradient of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile and (Solvent B) was used at a flow rate of 2.0 µL/min. The solvent B gradient began at 5% from 0 to 5 min, then 45% at 95 min, 80% at 95.1–96.1 min and 5% from 96.2 to 120 min. Peptides were searched using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version 27, rev. 11) without restricting species. Iodoacetamide derivative of cysteine was set as a fixed modification and oxidation of methionine was set as a variable modification. The +0.98 Da mass shift of asparagine was set as a variable modification in the analysis of both the tryptic peptides and the PNGase F peptides. Asn deamidated peptides in the tryptic (nondeglycosylated) dataset were considered false positives (Palmisano et al. 2012). Scaffold (version 3.00.08, Proteome Software Inc., Portland, OR) was used to analyze the MS/MS data. Only sites at an N-glycosylation sequence, N-X-[S/T], where X≠P is reported. Protein identifications with a minimum of two unique unmodified trypsin peptides and a probability ≥0.99 as per the Protein Prophet algorithm were considered significant (Nesvizhskii et al. 2003). Peptides were identified with a probability of ≥0.95 using the Peptide Prophet algorithm (Keller et al. 2002).

Supplementary Material

Supplementary Data
supp_25_4_403__index.html (1.3KB, html)

Acknowledgements

We thank Adrian Salic (Harvard Medical School, Cambridge, MA) for providing the propargylcholine used in this study, Rebekah Woolsey and David Quilici (Nevada Proteomics Center) for mass spectrometry analysis, Cynthia Mastick, David Shintani and Kathleen Schegg for helpful discussions, Grant Mastick, Tom Kidd and Scott Clark for comments on the manuscript.

Supplementary data

Supplementary data for this article are available online at http://glycob.oxfordjournals.org/.

Funding

This work was supported by the National Institutes of Health (RR-016464 from the INBRE Program of the National Center for Research Resources and RR024210/GM103554 from the COBRE Program of the National Center for Research Resources) and an IBRAF grant through the NSHE Health Sciences System.

Conflict of interest statement

None declared.

Abbreviations

ASP-6, aspartyl protease 6; FBS, fetal bovine serum; GalNAc, N-acetylgalactosamine; GalNAz, peracetylated azido-GalNAc; GlcNAc, N-acetylglucosamine; PC, phosphorylcholine; PC-proteins, PC-modified proteins.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_25_4_403__index.html (1.3KB, html)
supp_cwu122_cwu122supp.pdf (429.6KB, pdf)
supp_cwu122_cwu122supp_table1.pdf (26.4MB, pdf)

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