Glycobiology Advance Access originally published online on July 19, 2006
Glycobiology 2006 16(11):1052-1063; doi:10.1093/glycob/cwl024
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Oligosaccharides modulate the apoptotic activity of glycodelin
2 Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India; and 3 Glycobiology Institute, Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK
1 To whom correspondence should be addressed; e-mail: anjali{at}biochem.iisc.ernet.in
Received on March 31, 2006; revised on July 10, 2006; accepted on July 11, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm–zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.
Key words: apoptosis / glycans / glycodelin / mannosidase / sialic acids
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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GlycodelinA (GdA) is a glycosylated, dimeric, 162-amino acid protein secreted by human endometrium in the late secretory phase of the menstrual cycle and in the early phase of pregnancy under the regulation of the hormone, progesterone (Seppala et al., 1997
). Glycodelin (Gd) is glycosylated at two of the three putative glycosylation sites at asparagine residues N28 and N63 (Dell et al., 1995). Observations by several research groups suggest that GdA is immunosuppressive (Bolton et al., 1987), and earlier studies from our laboratory indicate that this effect is due to its capacity to induce apoptosis in activated T cells (Mukhopadhyay et al., 2001). Interestingly, another isoform of GdA synthesized by the seminal vesicles and secreted into the seminal plasma, glycodelinS (GdS), is not apoptogenic (Mukhopadhyay et al., 2004) even though both the isoforms share the same amino acid sequence. On the basis of our studies with glycosylation mutants of Gd expressed in Sf21 (Spodoptera frugiperda) insect cell line, using a baculoviral system, we have reported recently that the apoptogenic activity of Gd resides in its protein backbone, whereas the oligosaccharides regulate the manifestation of this activity (Jayachandran et al., 2004). The same Gd gene expressed in another insect cell line Tni, from Trichoplusia ni, failed to be apoptogenic, and the reason appeared to be due to differences in the composition of the glycans. Using mannosidase digestion and glycan profiling of the baculoviral system-expressed Gd proteins expressed in the two different insect cells, we show that the manifestation of the apoptogenic activity is unhindered by the presence of short glycan structures. We also observed that glycosylation of either one of the two glycosylation sites on Gd, with glycan chains comprising >5–6 monomeric units (two N-acetyl glucosamine and 3–4 mannose residues with or without a proximal fucose), is sufficient to mask the apoptogenic region. |
Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Molecular characterization of recombinant Gd
The molecular weights (MWs) of all the recombinant proteins expressed in insect cells were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and identity confirmed by western blot using the anti-Gd mAb, B1C2. We have reported previously (Jayachandran et al., 2004
) that the wild-type (WT) Gd-Sf21 separated as four distinct molecular species between MW 
26 and 20 kDa (Figure 1A). The WT Gd-Tni produced a diffuse band running between 18 and 28 kDa (Figure 1A). The N28Q and N63Q Gd-Tni resolved into molecular species running between 18 and 24 kDa. Gd-Chinese hamster ovary (CHO) electrophoresed as a single band of MW 28 kDa (Figure 1B). There was minimal mobility shift of the WT Gd-Tni protein as seen on 15% SDS–PAGE after mannosidase digestion (data not shown). On the contrary, GdS (Figure 1A) and Gd-CHO (Figure 1B) showed appreciably faster mobility following mannosidase digestion. Insect cells have been shown previously to produce paucimannosidic glycans (mostly trimannosyl core plus or minus fucose; Kuroda et al., 1990), whereas CHO cells produce predominantly complex type biantennary glycans and oligomannose type glycans (Hooker et al., 1999). 
1–2,3-Mannosidase will remove few mannose residues from the paucimannosidic glycans, which are unlikely to produce a discernible difference in SDS–PAGE profile. With the oligomannose-type glycans of Gd-CHO, the mobility difference is discernible, because more residues are removed.
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Glycan profiling reveals differences in glycosylation between Gd-Sf21 and Gd-Tni
The normal phase high-performance liquid chromatography (NPHPLC) chromatograms of the glycans released from the gel areas confirmed by western blotting revealed that the glycan composition of the two cell lines is the same but that the proportions are markedly different. Peptide-N-glycanase F (PNGaseF) is able to release glycans with 1-6 core fucose only. As it is known that insect cell lines can also produce glycans with 1-3 core fucose (Fabini et al., 2001; Morais et al., 2001), glycans were also released by ammonia-based ß-elimination. The same glycan assignments were achieved by this method as with PNGaseF, showing that there was no 1-3 core fucose and that all the glycans had been released from the gels (data not shown). The HPLC glycan profiles of the WT Gd, N28Q Gd and N63Q Gd proteins revealed both mannosylated and complex glycans (Figure 2A, Table I). The structures of the glycans were determined by a combination of exoglycosidase digests of the 2AB-labeled compounds, weak anion exchange (WAX) fractionation, and subsequent NPHPLC profiling and both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization mass spectrometry (ESI-MS) of the released glycans. A major difference between the glycan pools is in the percentage of core fucosylated trimannosylated glycan (FM3). The glycan pool from Gd-Sf21 (Figure 2Ai) contained 30% of FM3, whereas that from Gd-Tni (Figure 2Aiv) contained only 9% of FM3. Also Gd-Sf21 had 20% of Man5–7GlcNAc2 (M5–7) and 10% of the sialylated (S) triantennary (A3) trigalactosylated (G3) glycan, whereas the glycan pool from Gd-Tni had 26% of M5–7 and 19% of the same A3G3 glycans. When the glycans from mutant Gd-Sf21 (Figure 2Aii and iii) and Gd-Tni (Figure 2Av and vi) cell lines were analyzed, the different composition of the glycans at each of the occupied sites became clear. In both sets of NPHPLC profiles, it can be seen that the predominant glycan contained in the N28Q Gd is FM3, 56% of Sf21, and 21% of Tni total glycans. With N28Q Gd-Tni, however, there were also 60% of triantennary A3-sialylated structures. With N63Q Gd-Sf21, the predominant glycans at N28 are M3 (26%), with 23% of larger oligomannose (>M3) glycans. N63Q Gd-Tni, however, has only 5% of M3 and mostly larger oligomannose glycans (36%) and triantennary A3 structures (25%) (Figure 2A, Table I). The glycosylated sites of Gd-Sf21, therefore, contain mostly trimannose M3 glycans with and without fucose, whereas those of Gd-Tni contain mostly FM3 at N63 and M6–M8 glycans at N28.
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Figure 2Bi shows the complete glycan pool released from WT Gd-Tni protein. Figure 2Bii shows an NPHPLC trace of the jack bean -mannosidase (JBM) digest of the complete glycan pool showing the presence of oligomannose-type glycans. Figure 2Biii is of the 2–3,2,6 sialidase (Arthrobacter ureafaciens sialidase [ABS]) digest of the same sample showing the presence of sialylated structures. The WT FM3 glycan was also examined by negative ion nanospray MS, which clearly showed that the fucose was attached to the sixth position of the reducing-terminal GlcNAc residue (Harvey, 2005a,b). Structures of the high-mannose glycans were confirmed by their HPLC retention times and negative ion ESI-MS/MS. The latter technique showed that M6, M7, and M8 existed as the d3, d1, and d3 isomers, respectively (Rouse et al., 1998). M5 and M9 do not normally exist in isomeric forms. Structural representations of M6, A3G3S3, and A3G3 are shown in Figure 2B.
To elucidate the sialic acid linkages of the sialylated glycans, the complete glycan pool was digested with both ABS and 2–3 sialidase (NAN1). Both linkages were found (Table I). The complete glycan pools were also run on WAX HPLC, and the neutral and charged fractions, with 1–4 charges, were collected. These fractions were then run on NPHPLC both before and after digestion with the two sialidases. The complex sections of the profiles of the undigested fractions are shown in Figure 2C together with the complete complex glycan pool. After sialidase digestions, the non-sialylated core glycans were identified as A3G3 (a structure with a branched 3-antenna as determined by negative ion MS/MS; Harvey et al., 2005a,b), A3G2, and A2G2 (data not shown). There were at least 15 sialylated glycans, some of which also carry negative charges that were additional to those from the sialic acid residues. Following ABS digestion of the complete glycan pool, the glycans were again run on WAX HPLC when two small charged peaks remained (data not shown) confirming the presence of non-sialylated charged structures. Liquid chromatography (LC)–MS, which was performed on desialylated glycans from both Gd-Sf21 and Gd-Tni from the ß-elimination release, confirmed the presence of both complex and mannosylated glycans with and without an extra mass of 80 Da (Table I). This could be due to either a sulfate or a phosphate group. Sulfates, however, have previously been detected only as sodium adducts of which there were none in this spectrum (Harvey and Bousfield, 2005). Therefore, these glycans have been assigned as phosphated mannose. The presence of small quantities of M8 and M9, in addition to the other oligomannose sugars, was discernible in the glycans released from the Gd-Tni gel bands and also in the N63Q Gd-Tni. The results of both LC-MS and MALDI MS are summarized in Table I.
Previous studies have mainly shown that insect cell lines produce glycoproteins with oligomannose and core fucosylated trimannose only (Kuroda et al., 1990) with as much as 75% of the glycans being paucimannose with and without core fucosylation (Kulakosky et al., 1998). Other studies, however, have reported that baculovirus infection of lepidopteran insect cell line Sf21 makes available enzymes that provide and utilize the substrate M5-GlcNAc2 protein. This allows the production of glycans with increasing proportions of complex sugars with and without sialylation as the time of incubation increases, with up to 96% complex glycans after 90 h incubation time (Davidson et al., 1990; Velardo et al., 1993). The baculovirus-infected cell lines in this study were incubated for 120 h; however, although complex glycans were produced, the greater proportion, 85% for Gd-Sf21 and 60% for Gd-Tni, remained as mannose-type glycans.
Inhibition of Jurkat cell proliferation by recombinant Gd
The WT Gd-Sf21 and the WT Gd-Tni along with Tni-expressed glycosylation mutants N28Q Gd-Tni, N63Q Gd-Tni, and N28Q,N63Q Gd-Tni (double mutant) were tested for the inhibition of Jurkat cell proliferation. Although the WT Gd-Tni and the N63Q Gd-Tni exhibited no activity, the N28Q Gd-Tni protein inhibited proliferation, though with an efficiency lower than that of WT Gd-Sf21 (Figure 3, Tables II and III). The IC50 was found to be around 60–70 nM as against an IC50 of 40–50 nM seen with WT Gd-Sf21 (Tables II and III). The purified Gd-CHO did not inhibit the proliferation of Jurkat cells with the concentrations tested (Figure 3). The counts per minute values of measured radioactivity are plotted as percentage incorporation of tritiated thymidine for comparison amongst the different proteins.
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Gd-Tni and Gd-CHO inhibit proliferation of Jurkat cells following mannosidase digestion
The WT Gd-Tni failed to inhibit the proliferation of Jurkat cells. However, after mannosidase digestion, it exhibited activity comparable with that of WT Gd-Sf21 with an IC50 value of 45–55 nM (Figure 3, Tables II and III). Before mannosidase digestion, N28Q Gd-Tni showed IC50 value of 60–70 nM, whereas the N63Q Gd-Tni showed no activity even at 100 nM concentration. After mannosidase digestion, both N28Q Gd-Tni and N63Q Gd-Tni showed an increase in activity, with N28Q Gd-Tni showing an IC50 of 25–30 nM, whereas N63Q Gd-Tni showed an IC50 of 45–55 nM. The Tni double mutant showed an IC50 value of 30–40 nM similar to that of Sf21 double mutant (Figure 3, Tables II and III). On mannosidase digestion, Gd-CHO also showed activity with an IC50 value of 125 nM.
GdS does not show activity even after mannosidase treatment
As has been reported earlier (Mukhopadhyay et al., 2004), GdS did not inhibit Jurkat cell proliferation even at a 20-fold higher protein concentration. Even after mannosidase digestion, GdS did not manifest inhibitory activity (Figure 3). No apoptotic population was observed in Jurkat cells treated with GdS, both in fluorescence-assisted cell sorter (FACS) analysis and in the ethidium bromide–acridine orange staining procedure (Figure 4).
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Apoptotic assays confirm the Jurkat cell proliferation assay results
Jurkat cells cultured with WT Gd-Tni or the glycosylation mutant proteins, which had been earlier digested with 1–2,3-mannosidase or treated with buffer alone as control, were stained with ethidium bromide and subjected to FACScan analysis. The apoptotic population was identified by the appearance of sub-G0/G1 peak (Mukhopadhyay et al., 2001). Treatment of Jurkat cells, with the recombinant proteins WT Gd-Tni and N63Q Gd-Tni without mannosidase digestion, showed no apoptotic population. Following mannosidase treatment, the above proteins showed an increase in the apoptotic population from 10 to 45–55% (Table III). The only exception was N28Q Gd-Tni, which showed 25% apoptotic population before mannosidase digestion and this increased to 45% after mannosidase digestion. Tni double mutant was apoptogenic even in the absence of mannosidase digestion (Figure 4, Table III).
Changes in the nuclear morphology characteristic of apoptosis were observed in Jurkat cells by dual staining with ethidium bromide and acridine orange (Mukhopadhyay et al., 2001). On co-culturing Jurkat cells with various recombinant Gd proteins, the cells underwent nuclear fragmentation and blebbing with N28Q Gd-Tni and N28Q,N63Q Gd-Tni (double mutant) proteins in the absence of mannosidase digestion (Figure 4). After mannosidase digestion, WT Gd-Tni and N63Q Gd-Tni exhibited apoptogenic activity. GdS failed to manifest apoptogenic activity even on mannosidase digestion. Although Gd-CHO did not induce any apoptosis, upon mannosidase digestion, it exhibited apoptotic activity with an increase in percentage of cells undergoing apoptosis from 7 to 31% (Table III).
WT Gd-Sf21 expressed in the presence of deoxy-nojirimycin shows less apoptotic activity
Deoxy-nojirimycin (DNJ Gd), an inhibitor of glucosidase I and II, was used to express WT Gd-Sf21 with larger glycan structures in Sf21 cells. The western blot shows reduced mobility of the DNJ Gd (Figure 5A) because of the presence of glucosylated M9 glycan form. This protein showed a higher IC50 value of 150 nM when compared with the untreated WT Gd-Sf21 (Figure 5B).
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Tni cell line gives better expression of Gd proteins
Competitive enzyme-linked immunosorbent assay (ELISA) with the culture supernatants of Tni and Sf21 cell lines infected with various recombinant baculoviruses shows that Tni cells yielded a higher protein expression than Sf21 cells (Table IV). The fold difference seen between Tni- and Sf21-expressed proteins becomes narrower with N28Q Gd and N28Q,N63Q Gd (double mutant) proteins. Transfected CHO cells expressed 10–12 mg of Gd-CHO per liter of culture medium.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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We previously showed that the glycans of GdA modulate the apoptogenic activity of the molecule (Jayachandran et al., 2004
) and also that desialylation resulted in a decrease in the apoptogenic activity (Mukhopadhyay et al., 2004). We hypothesized then that the presence of sialic acid might open up the apoptogenic region. It was, therefore, surprising when the recombinant Gd expressed in the Sf21 insect cells demonstrated apoptogenic activity, as the recombinant proteins from insect cells are reported to carry paucimannose glycan structures (Jenkins and Curling, 1994). We further hypothesized therefore that the small glycan structures on the glycoproteins expressed in Sf21 cells did not hinder the accessibility of the apoptogenic region of Gd. The Tni cell line, which is known to give a higher protein expression in a baculoviral system, was used later to express larger amounts of Gd proteins for the study. Interestingly, the WT Gd-Tni was found to be inactive even though it would potentially carry the same glycans. Glycan analysis of recombinant Gd proteins was, therefore, undertaken to elucidate the possible differences.
On analyses of the total pool of glycans from in-gel release, it was observed that the predominant species in the WT Gd-Sf21 was FM3 (Table I). The percentage of glycans with FM3 structure or smaller was found to be >50% (Table I). Glycan analyses of Gd-Sf21 mutant proteins have shown it to contain predominantly FM3 at site N63 and M3 at site N28. With WT Gd-Tni glycans, FM3 and smaller forms constituted 27%. Again analyses of glycans from Gd-Tni mutant proteins showed it to contain predominantly FM3 at N63 but M6 at N28. In the case of the WT Gd-Tni, N28 could be predominantly occupied by larger mannosylated or triantennary glycans, which could partially or completely cover the apoptogenic region. We carried out a mannosidase digestion of the WT Gd-Tni protein, which resulted in the manifestation of the apoptogenic property comparable with that of WT Gd-Sf21, corroborating our hypothesis.
GdS has been reported to carry a mannose-rich glycan structure at position N28 and a complex glycan structure, which is non-sialylated and heavily fucosylated, at position N63 (Morris et al., 1996). GdS failed to induce apoptosis even after treatment with mannosidase (Figure 4) or with EndoH digestion of GdS (Mukhopadhyay et al., 2004). This observation is consistent with the presence of a complex glycan structure at position N63 that can hinder the accessibility of the apoptogenic region even in the absence of a paucimannose N28 glycan structure.
Our observations that N63Q Gd-Tni is not apoptogenic but manifests this activity after mannosidase digestion suggest that the presence of larger oligomannose glycans (40% M5–M9), at position N28, confirmed here by glycan analysis is inhibitory to apoptogenic activity. N28Q Gd-Tni is apoptogenic, albeit at a higher concentration (IC50 of 70 nM) (Table II), without mannosidase digestion, which may be due to the 21% occupancy of N63 by FM3.
The activity data obtained with the different forms of the recombinant Gd expressed in insect cells are shown in Figure 6. The accessibility to the apoptogenic area is masked to different extents by N-linked glycans at sites N28 and N63. Larger glycans on N28, but not on N63, mask the apoptogenic region on Gd resulting in inactive protein, as is the case with WT Gd-Tni and Gd-Tni N63Q. Clipping these chains as achieved by mannosidase digestion makes the apoptogenic region accessible, thus imparting apoptotic activity to the molecules. The presence of complex glycans as in case of GdS, however, is different from the simple glycan chains of the recombinant Gd. In the case of the mannosidase-digested GdS, the apoptogenic activity is still masked by heavily fucosylated glycans at N63 (Figure 6). The WT Gd-Sf21 expressed in the presence of DNJ Gd shows reduced activity (Figure 5) further supporting our hypothesis that larger glycans mask the apoptogenic region.
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Interestingly, Gd-CHO, which did not display apoptogenic activity on Jurkat cells, became active after mannosidase treatment (Figure 4, Tables II and III). The CHO cell line is known to add complex glycans and a high proportion (>60%) of oligomannose glycans (Matsuura et al., 1998), which would be cleaved to the FM2 form. It was earlier shown that Gd expressed using CHO cells carry typical CHO-type glycans, the major N-glycans present being the lacNAc-based complex type glycans devoid of any lacdiNAc glycans as normally seen in GdA (Van-den-Nieuwenhof et al., 2000). Moreover, the CHO-expressed Gd did not bind Wisteria Floribunda lectin (WFA) and Sambucus nigra lectin (SNA) blots, demonstrating the absence of ß-linked GalNAc residues and terminal NeuNAc2–6Gal(NAc) sequences, respectively. These earlier reports further provide evidence for the Gd-CHO to be inactive unless subjected to mannosidase digestion.
The expression of glycoproteins containing complex glycans in insect cells has been much debated, especially for their capacity to sialylate recombinant proteins (Davidson et al., 1990; Kuroda et al., 1990; Velardo et al., 1993; Kulakosky et al., 1998; Kost et al., 2005). Our work demonstrates that both the Sf21 and Tni cell lines do add sialic acid residues to recombinant proteins although at a lower percentage than reported elsewhere.
Our studies confirm that in the case of Gd, the glycan chains exert their effect by modulating the manifestation of the apoptogenic property of the amino acid backbone by masking its expression in the case of male reproductive system isoform GdS, while permitting the same in the female reproductive system isoform GdA. In the physiological context, it is not the absence of glycosylation but it is the presence of complex glycans carrying terminal sialic acid residues at sites N28 and N63 of GdA that result in unmasking of the cryptic apoptogenic region. It must be noted here that 60% of all the glycans from GdA, but none from GdS, have been shown to contain at least one antenna terminating in sialic acid (Dell et al., 1995). The unmasking of the apoptogenic region of GdA by sialylated structures could possibly be due to charge-based interactions with the T-cell surface receptors to which Gd might bind, with complementary-charged regions pulling the glycans away from the binding site. This mechanism would not occur with uncharged glycans, leaving the size of the neutral glycan structures being the only factor determining the accessibility of the apoptogenic region.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Cells and cell lines
The Sf21 cells (S. frugiperda) (Invitrogen) and Tni5B1–4 cells (T. ni) (Invitrogen, Carlsbad, CA) were maintained in TC 100 medium (Sigma) and TNM-FH medium (Sigma), respectively, supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO) and 50 µg/mL of gentamycin sulfate at 27°C. Cell cultures were maintained as either monolayers in Nunc T-75-cm2 flasks or shaker culture in 250-mL Schott conical flasks in the presence of 0.1% pluronic F68 (Sigma) and passaged every 5 days.
Jurkat (JR4) human T cell line was cultured in RPMI 1640 medium (Sigma), and CHO cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma), both supplemented with 10% FBS at 37°C in a 5% CO2 incubator and passaged twice a week.
Cloning the Gd cDNA into baculoviral system
The Bac-to-Bac baculovirus expression system (Invitrogen) was used to clone the Gd cDNA. In brief, the full-length Gd cDNA with the secretory signal, obtained from the uterine endometrium, was cloned into the pFASTBAC1 vector. The recombinant vector, on transformation into the Escherichia coli strain DH10BAC, generates recombinant bacmid, which on lipofection into insect cells results in the production of recombinant virus, which produces the protein of interest. The N-linked glycosylation mutants of Gd, namely N28Q Gd, N63Q Gd, and the double mutant N28Q,N63Q Gd, were generated as described previously (Jayachandran et al., 2004).
Cloning and expression of Gd in CHO cell line
The Gd gene along with its secretory signal had been cloned earlier into the vector pcDNA3 (Invitrogen), in our laboratory, between sites BamH1 and Pst1 using RT–PCR from RNA isolated from the uterine endometrium. Stable transfection of the vector into CHO cells was done using LipofectAMINE reagent (Invitrogen) in opti-MEM medium (Gibco-BRL, Carlsbad, CA). The clones were selected for G418 (Sigma) resistance of up to 1 mg/mL concentration in the culture medium. The cells were cloned to monoclonality by the limiting dilution method in a 96-well plate (NUNC, Rochester, NY). Clones secreting maximal amount of recombinant Gd (Gd-CHO) were selected by ELISA using anti-Gd-specific mAb, D9D4, as described later in the text.
Purification of the recombinant Gd proteins
Because of the presence of the mammalian secretory signal, Gd was secreted into the culture medium. It was purified by passing the culture supernatant through a Gd-specific mAb (D9D4) immunoaffnity column. The column was washed with phosphate-buffered saline (PBS) (50 mM phosphate buffer, pH 7.2, containing 150 mM NaCl) and the bound protein eluted with 100 mM glycine–HCl, pH 2.5. Collected fractions were immediately neutralized with 1 M Tris, pH 8.0, and the fractions containing protein were pooled and concentrated using a centricon concentrator (Vivascience, Goettingen, Germany). The concentrate was dialyzed overnight against PBS, filter-sterilized through a 0.45-µm membrane filter (Sartorius, Goettingen, Germany), and the protein concentration was estimated by Stauffer’s modification of Lowry’s method (Stauffer, 1975).
Quantitation of the secreted Gd by ELISA
The insect cells were seeded at a density of 20 x 106 cells per 10 mL of culture medium and were infected with the recombinant virus with a multiplicity of infection of 10. After 96 h, the cells were spun down, and the culture supernatants were assayed for the concentration of Gd by competitive ELISA. Briefly, 100 µL of a solution of recombinant E. coli-expressed Gd at a concentration of 10 µg/mL in PBS was coated on to wells of an ELISA plate, and unoccupied sites were blocked with radio-immuno assay (RIA) buffer (0.2% bovine serum albumin [BSA] in PBS). Varying dilutions of the insect cell culture supernatant, recombinant Gd-CHO-containing spent medium, or the standard GdA were then incubated with appropriately diluted Gd-specific-mAb D9D4 (at a dilution of the antibody exhibiting 80% of maximum binding). The binding of the mAb to immobilized antigen was determined by subsequent incubation with horse-radish peroxidase-conjugated rabbit secondary antibody to mouse immunoglobulin (Dakopatts, Glostrup, Denmark), followed by incubation with the substrate tetra-methyl-benzidine (TMB) (Bangalore Genie, Bangalore, India). The absorption was read at 450 nM and the concentration of recombinant Gd protein determined from the standard graph plotted using GdA.
Mannosidase digestion of Gd
Mannosidase digestion was carried out using the 1–2,3-mannosidase (New England Biolabs, Ipswich, MA) as per the manufacturer’s protocol. In brief, 10 µg of the purified and filter-sterilized protein was treated with 2 U of the enzyme for a period of 12 h with the G3 buffer provided with the enzyme, in a 20-µL reaction mixture at 37°C. The treated protein was analyzed by SDS–PAGE followed by Coomassie blue staining and western blotting. For western blotting, the proteins were transferred onto nitrocellulose membrane and probed with Gd-specific-mAb B1C2 (Karri et al., 2000). The mannosidase-treated protein was used in Jurkat cell proliferation and apoptosis assays for testing its activity.
Analysis of glycans released from Gd-Bac
Twelve micrograms each of WT Gd-Sf21 and WT Gd-Tni proteins was electrophoresed on 15% polyacrylamide gels under reducing conditions in a vertical mini-gel system, a modification (Radcliffe et al., 2002) of the method described by Küster and others (1997). Gel sections equivalent to the areas that were confirmed by western blotting with Gd-specific mAb B1C2 were excised. These sections contain the total protein, which was used in the apoptotic assays. Glycans from Gd glycosylation mutants N28Q and N63Q expressed in the Sf21 and Tni cell lines were also analyzed. Glycans were also released by ammonia-based ß-elimination from 50 µg of samples (Huang et al., 2002) to detect the presence of any sugars resistant to digestion by PNGaseF (EC 3.5.1.52
[EC]
, Roche Diagnostics, Mannheim, Germany). The extracted glycans were labeled with the fluorophore 2-aminobenzamide (2AB) by reductive amination (Bigge et al., 1995) and processed through NPHPLC, using a low salt buffer system (Guile et al., 1996). The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to provide a dextran ladder from which the retention times for the individual glycans were converted to glucose units (GU). These GUs were compared with a database of experimental values to obtain preliminary assignments for the glycans that were confirmed by digestions with exoglycosidases and MS, both MALDI and LC/ESI-MS. Further analysis of the glycans was carried out using WAX HPLC (Royle et al., 2002).
Exoglycosidase digestions
Exoglycosidase digestions were carried out on the 2AB-labeled glycan pools of the N-linked glycans, and terminal residues were released from the nonreducing terminus using appropriate arrays of enzymes: ABS (EC 3.2.1.18
[EC]
) releases 2–6- and 2–3-linked sialic acids, Streptococcus pneumonia sialidase recombinant from E. coli (NAN1, EC 3.2.1.18
[EC]
) releases only 2–3-linked sialic acids, bovine testes ß-galactosidase (BTG [EC 3.2.1.23
[EC]
]) releases galactose with ß1–3 and ß1–4 linkages, ß-N-acetyl-glucosaminidase cloned from S. pneumonia, expressed in E. coli (GUH [EC 3.2.1.30
[EC]
]), releases N-acetylglucosamine (GlcNAc) with ß1–2,3,4 and 6 linkages, bovine kidney fucosidase (BKF [EC 3.2.1.51
[EC]
]) releases 1–6,2,3- and 4-linked fucose, and JBM (EC 3.2.1.24
[EC]
) releases 1–2,3- and 6-linked mannose. Incubations were for 16 h at 37°C, pH 5.5, using 50 mM sodium acetate buffer, pH 5.5, or in 100mM sodium acetate, 2 mM Zn2+, pH 5.0, for the JBM digestion. All enzymes are available from Prozyme, San Leandro, CA.
Glycan analysis by LC/ESI-MS
Glycans were analyzed using an LC Packings Ultimate HPLC equipped with a Famos autosampler (Dionex, Leeds, UK) interfaced with a Q-Tof Ultima Global mass spectrometer (Waters-Micromass, Manchester, UK). Chromatographic separation was achieved using a 2 x 250 mm, microbore NPHPLC TSK gel Amide-80 column (Hichrome Ltd., Theale, UK) with the same gradient and solvents as used with the standard NPHPLC but at a lower flow rate of 40 µL/min. The mass spectrometer was operated in positive ion mode with 3 kV capillary voltage, RF lens 60, source temperature 100°C, desolvation temperature 150°C, cone gas flow 50 L/h, and desolvation gas flow 450 L/h (Royle et al., 2002).
MALDI-TOF-MS
Positive ion reflectron-MALDI-time-of-flight (TOF) mass spectra were recorded with a Waters-Micromass TofSpec 2E reflectron mass spectrometer (Waters-Micromass) fitted with delayed extraction and a nitrogen laser (337 nm). The acceleration voltage was 20 kV, the pulse voltage was 3200 V, and the delay for the delayed extraction ion source was 500 ns. Samples were prepared by adding 0.5 µL of an aqueous solution of the sample to the matrix solution (0.3 mL of a saturated solution of 2,5-dihydroxybenzoic acid [DHB] in acetonitrile) on the stainless steel target plate, allowing it to dry at room temperature and then recrystallizing it from ethanol (Harvey, 1993).
Negative ion nanospray MS
Negative ion collision-induced dissociation spectra of selected glycans were obtained with a Waters-Micromass Ultima Global quadrupole-TOF (Q-Tof) mass spectrometer fitted with a nanospray ion source. Samples in water:methanol (1:1, v:v) were infused with Proxeon nanospray capillaries. Operating conditions were ion source temperature, –120°C; nanospray capillary potential, 1.2 kV, RF-1 potential, 180 V; cone voltage, 100 V; collision gas, argon; and collision cell voltage in the range 50–100 V (appropriate for the mass of the particular compounds). Spectral acquisition and processing were achieved with Mass-Lynx 4 software (Waters-Micromass).
Biological activity of recombinant Gd
Jurkat cell proliferation assay
Jurkat (JR4) cells 0.1 x 106/200 µL of RPMI 1640 medium supplemented with 10% FBS were cultured with varying concentrations of Gd for 24 h in a 96-well plate (NUNC) along with appropriate controls. Approximately 0.1 mCi of 3H-thymidine (methyl T) (Board of Radiation and Isotope Technology, Mumbai, India) was added after 24 h and incubated further for 6 h. The cells were harvested and lysed on glass fiber (GF/C) filters (Whatman, Middlesex, UK) using a cell harvester (NUNC). The filters were dried, and the 3H-thymidine incorporated was measured in a scintillation counter (Wallac, Wellesley, MA). All treatments were carried out in triplicates.
Apoptosis assay
Acridine orange–ethidium bromide staining was used: 1 x 106 Jurkat JR4 cells were cultured with Gd protein, before and after digestion with mannosidase, for 16 h in 500 µL of RPMI 1640. The cells were then harvested, centrifuged at 300 x g for 5 min, and resuspended in 25 µL of medium along with 1 µL of a dye mixture of acridine orange and ethidium bromide (100 µg/mL each in PBS). The cells were observed immediately under a fluorescence microscope (Carl Zeiss) using a blue filter.
FACS analysis of Gd-Bac-treated Jurkat cells
Jurkat JR4 cells were plated at a density of 1 x 106/500 µL of RPMI supplemented with 10% FBS and incubated for 12 h with Gd or control proteins. The cells were harvested, washed with PBS, and fixed in 70% ethanol for 1 h. After a wash with RIA buffer (0.2% BSA–PBS), the cells were resuspended in 250 µL of ethidium bromide staining solution (50 µg/mL ethidium bromide, 100 µM ethylenediaminetetraacetic acid [EDTA], and 50 µg/mL ribonuclease A in PBS) for 1 h at 45°C and analyzed by FACScan (Becton and Dickinson, Franklin Lakes, NJ) using blue light.
Expression of WT Gd-Sf21 in the presence of DNJ
Sf21 insect cells were seeded at a density of 20 x 106 cells per 10 mL of culture medium and were infected with the recombinant WT Gd-Bac baculovirus with a multiplicity of infection of 10. At the end of 48 h, DNJ Gd, an inhibitor of glucosidase (Steiner et al., 2004) was added to the culture medium to a final concentration of 2.5 mM. The cells were further incubated for 72 h, at the end of which the culture supernatant was harvested and WT Gd-Sf21 protein purified by immunoaffnity column. The purified protein was then assayed for its activity on the proliferation of Jurkat cells.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The authors gratefully acknowledge the Department of Biotechnology (DBT), Government of India, for the financial support. The glycan analysis was funded by the Oxford Glycobiology Institute Endowment, and the MS analysis was supported by grants from BBSRC and the Wellcome Trust. We thank Dr Omana Joy for FACS analysis.
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Abbreviations |
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AB, aminobenzamide; ABS, Arthrobacter ureafaciens sialidase; DNJ, deoxy-nojirimycin; ELISA, enzyme-linked immunosorbent assay; ESI-MS, electrospray ionization mass spectrometry; FACS, fluorescence-assisted cell sorter; FBS, fetal bovine serum; FM3, fucosylated trimannosylated glycan; Gd, Glycodelin; GdA, GlycodelinA; Gd-CHO, Chinese hamster ovary-expressed glycodelin; GdS, GlycodelinS; Gd-Sf21 and Gd-Tni, baculoviral system expressed glycodelin; GU, glucose units; JBM, jack bean
-mannosidase; LC, liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MW, molecular weight; NPHPLC, normal phase high-performance liquid chromatography; PBS, phosphate-buffered saline; PNGaseF, peptide-N-glycosidase F; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Sf21, Spodoptera frugiperda; Tni, Trichoplusia ni; WAX, weak anion exchange; wild type, WT |
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