Glycobiology Advance Access originally published online on October 19, 2007
Glycobiology 2008 18(1):42-52; doi:10.1093/glycob/cwm113
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A Tetraantennary Glycan with Bisecting N-Acetylglucosamine and the Sda Antigen is the Predominant N-Glycan on Bovine Pregnancy-Associated Glycoproteins
3 Abteilung Neuroanatomie, Medizinische Hochschule Hannover, 30625 Hannover, Germany
4 Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College, London SW7 2AZ, UK
5 Abteilung für Toxikologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany
6 Klinik für Geburtshilfe, Gynäkologie und Andrologie der Groß- und Kleintiere, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany
7 Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark
8 Institut für Veterinäranatomie, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany
2 To whom correspondence should be addressed. Tel: +44 11595 16464 Fax: +44 11595 16415; e-mail: karl.klisch{at}nottingham.ac.uk
Received on May 21, 2007; revised on September 14, 2007; accepted on October 1, 2007
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Abstract |
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Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sda-antigen (NeuAc
2-3[GalNAcβ1-4]Galβ1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sda-antigen, shows that BNC granules contain the Sda-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to 2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy. Key words: cattle / gestation / glycosylation / mass spectrometry / placenta
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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The fetal binucleate trophoblast giant cells (BNCs) in the ruminant placenta produce several glycoproteins and store these proteins in cytoplasmic granules. Mature BNCs fuse with maternal uterine epithelial cells, exocytose the granules at the basal membrane of the uterine epithelium, and thereby deliver the proteins into the maternal organism (Wooding 1992
). This process happens continuously from the onset of BNC-formation in the third week of gestation until parturition. Among the cargo-proteins of the granules are pregnancy-associated glycoproteins (PAGs), which belong to the protein family of aspartic proteinases. In the evolution of ruminants, gene duplications led to a high number of PAG-genes (
100) in cattle and sheep (Xie et al. 1997). The high ratio of nonsynonymous to synonymous mutations in the evolution of PAG sequences led to the suggestion that natural selection caused the diversification of ruminant PAGs (Xie et al. 1997). This suggests that the diversity of PAGs is of functional importance; but the function of PAGs is still not well characterized.
Lectin histochemical studies show that BNC granules have a very specific glycosylation pattern (Munson et al. 1989; Lehmann et al. 1992; Jones et al. 1994; Nakano et al. 2002; Klisch and Leiser 2003). During most of the time of pregnancy, the granules can be labeled with Phaseolus vulgaris leucoagglutinin (PHA-L), which recognizes branched tri- and tetraantennary glycans with ß1-6 linked GlcNAc, and with Dolichos biflorus agglutinin (DBA), which binds to terminal N-acetylgalactosamine (GalNAc) residues. The main targets of these lectins in BNCs are PAGs and the GalNAc-binding lectins proved to be useful tools for affinity-chromatographic PAG purification (Klisch and Leiser 2003; Klisch et al. 2005). Before parturition, the glycosylation of PAG changes and terminal GalNAc residues are largely absent at term (Klisch et al. 2006).
This very specific pattern of PAG-glycosylation and its temporal changes during gestation strongly indicate a functional role of the carbohydrates during pregnancy. In the present study, we characterize the main N-glycans which are attached to PAGs and we further characterize the changes of glycosylation that occur in early pregnancy and at term.
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Results |
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MALDI-MS of native and desialylated PAG-glycans
Purification and characterization of PAGs was done as previously described (Klisch et al. 2005
). From the purified PAGs, the N-glycans were released by digestion with PNGase-F. The MALDI-time of flight (TOF) spectrum in native glycan preparations (Figure 1) was dominated by a molecular ion at m/z 4697.2. Treatment of the native samples with neuraminidase reduced the value to m/z 3556.2, which corresponds to a loss of four molecules of N-acetylneuraminic acid (NeuAc). It should be mentioned that we were able to desialylate the PAG-glycans only with the Clostridium perfringens neuraminidase from Roche (molecular weight (MW) 60 kDa), but not with C. perfringens neuraminidase from New England Biolabs (MW 41 kDa) (data not shown). The peak at 3847 in the neuraminidase treated samples results from incomplete desialylation, with one molecule of sialic acid left. These major compounds are core fucosylated tetraantennary structures with bisecting GlcNAc and terminal NeuAc2-3[GalNAcβ1-4]Galβ1-4GlcNAc (Sda)-epitopes on all four antennae as explained in the subsequent section. Minor compounds are the corresponding triantennary structures (m/z 3837.7, 2988.0 in the native and desialylated sample respectively) and their counterparts without core fucosylation (m/z 3690.8, 2841.3).
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MALDI-MS and MS/MS of permethylated PAG N-glycans
To corroborate the results obtained from MS analysis of native samples, and to facilitate unambiguous sequencing by MS/MS, the PAG glycans were permethylated and analyzed by MALDI-TOF and MALDI-TOF/TOF. The MALDI-TOF spectrum (Figure 2A) was dominated by a molecular ion at m/z 5812, which is the predicted value for the permethylated counterpart of m/z 4697 in the native sample. The second most abundant signal in the spectrum is m/z 1071, which is a fragment ion derived from the antennae (see annotation in Figure 2B). Several additional glycans are present but they are of low abundance. Thus, the cluster of weak signals near m/z 2000 are molecular ions for high mannose glycans (Man6–9 at m/z 1784, 1988, 2192, and 2396, respectively), and the minor signals between m/z 3500 and m/z 5000 correspond to bi- and triantennary glycans with varying levels of core fucosylation. For example, m/z 3702 and 4757 are bi- and triantennary analogues of m/z 5812, whilst m/z 3528 and 4583 are their nonfucosylated counterparts.
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The major component at m/z 5812 was subjected to collisional activation in a MALDI-TOF-TOF experiment (Figure 2B). The fragmentation pattern is dominated by cleavage of the antennae on the reducing side of GlcNAc to yield m/z 4743 (loss of the Sda-epitope from the molecular ion) and m/z 1071 (the Sda-epitope itself; m/z 1093 is a sodiated adduct). The signal at m/z 5437 corresponds to loss of sialic acid from the molecular ion.
Glycomics analysis of placental tissues
Permethylated N- and O-glycans from unfractionated placentomal tissue of one mid-pregnant (gd 220) and one ante partal (approximately one day before term) cows were analyzed. This experiment allows the comparison of the general glycosylation pattern with the glycosylation of PAGs and it also gives MS-data about the ante partal changes of glycosylation. The N-glycan spectra of gd 220 (Figure 3A and C) and preterm (Figure 3B and D) tissues were both dominated by high-mannose glycans and by core fucosylated bi-(m/z 2652), tri- (m/z 2897, 3305, 3550), and tetraantennary (m/z 3754, 3958, 4145) glycans with capping gal–gal residues. The high mannose glycans were also observed among the PAG N-glycans (see Figure 2), while the latter structures were completely absent in the PAG-glycans. The main difference between the two samples were the Sda-capped glycans (m/z 4583, 4757, 5812), which were only observed in the gd 220-sample, but not in the near-term sample. Also, the Sda fragment ion (m/z 1071) was only observed in the gd 220 sample.
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The analysis of permethylated O-glycans also revealed differences mainly concerning the presences of Sda-epitopes. A m/z 1501 ion was observed in the gd 220 (Figure 4A) sample and in the PAG-O-glycans (Figure 4C), but not in the prepartum sample (Figure 4B). The PAG-O-glycans were dominated by the Sda fragment ion (m/z 1071).
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Linkage analysis of permethylated PAG N-glycans
A sample of permethylated PAG-glycan was converted to a mixture of partially methylated alditol actetates which were subjected to GC-MS linkage analysis. The region of the total ion chromatogram that was especially informative is reproduced in Figure 5. One of the major peaks is 3,4-linked Gal which is derived from the Sda-epitope. The abundant 3,4,6-linked mannose (Man) peak provides evidence for bisecting GlcNAc, whilst the major peaks for 2,4- and 2,6-linked Man confirm the presence of tri- and/or tetraantennary glycans. The less abundant 3,6-Man peak is attributed to the minor high mannose population. The fact that terminal GalNAc is significantly more abundant than terminal GlcNAc corroborates the assignment of the Sda-antennae. The presence of a major peak for 4-GlcNAc, and the absence of 3-GlcNAc, defines type 2 antennae backbones (Gal1-4GalcNAc). Fucosylation of the core is confirmed by the 4,6-GlcNAc peak.
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Western analysis
The CT1 antibody reveals one major band at approximately 67 kDa and a minor band at 75 kDa in the non-N-deglycosylated samples (Figure 6). These bands are not present in the PNGase F treated samples. The PAG antiserum shows a generally more intensive staining with several bands in the glycosylated and deglycosylated samples. The two most intensive bands in the non-N-deglycosylated sample are of the same MW as in the CT1 staining and are also missing in the deglycosylated samples. In the prolactin related protein-I (PRP-I) staining, the PNGase F treatment results in a shift from approximately 37 kDa to 25 kDa.
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Histochemistry
PAG positive BNCs were detected in all studied stages of pregnancy (Figure 7). PHA-L is generally colocalized with PAG. In gd 20 and 23, all PAG-positive BNCs are also labeled with Maackia amurensis lectin (MAL), but not with CT1 (Figure 8). The staining pattern substantially changes after gd 30: there is strongly reduced MAL staining, but the granules become CT1-positive. The staining with DBA was mainly similar to that of CT1 and is therefore not shown. This staining pattern with CT1-positive or DBA-positive granules continues until a few days before parturition. An intermediate staining was observed in the samples which were taken approximately five days before parturition. In the term samples, BNCs were mainly CT1/DBA-negative and MAL-positive.
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PCR
The relative gene expression of the β4GalNAcT-II is shown in Figure 9. Due to the uneven distribution of the data obtained by Real Time RT-PCR, the Kruskal–Wallis Test (a nonparametric ANOVA) was applied with the statistical software program, GraphPad3 (GraphPad Software, Inc., San Diego, CA,). The Kruskal–Wallis Test (nonparametric ANOVA) considered the differences between the different stages of pregnancy as not significant (p = 0.3430).
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Discussion |
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This report shows that one carbohydrate structure is predominating on bovine PAGs in midpregnancy. This is a core-fucosylated tetraantennary glycan in which all antennae carry a terminal Sda-antigen (Figures 1–3 and 5). The occurrence of bisecting GlcNAc in tetra- and triantennary glycans is a highly unusual feature. Generally, the attachment of bisecting GlcNAc to the core β-mannosyl residue by the N-acetylglucosaminyltransferase-III (GlcNAc-TIII) blocks the initiation of a tri- and tetraantennary branching pattern by GlcNAc-TIV and GlcNAc-TV (Schachter 1986
). This usually results in a reduced branching of N-glycans in cells which express GlcNAc-TIII. A strictly regulated localization of these GlcNAc-transferases in the Golgi-cisternae could circumvent this, and could thereby be a precondition for the synthesis of highly branched glycans with a bisecting GlcNAc (Sasai et al. 2003). Such a sublocalization, in which GlcNAc-TIV and -TV are localized in earlier Golgi-subcompartments than GlcNAc-TIII, should facilitate the initiation of a tetra- and tri-antennary branching pattern before the bisecting GlcNAc is attached. In addition, GlcNAc-TIII itself is inhibited by the addition of galactose to the growing antennae by the ß-1,4-galactosyltransferase (Fukuta et al. 2000). This implies that GlcNAc-TIII should also be spatially well separated from ß-1,4-galactosyltransferase or must be abundantly expressed. A second unusual feature is the relative uniformity of the tetraantennary Sda-glycan on PAGs in BNCs. Since most other glycoproteins show a large variety of attached glycans, with dominating bi- and triantennary structures, the dominance of the tetraantennary PAG glycans points at a highly regulated glycosylation machinery in BNCs. An alternative explanation for this phenomenon could be a bias in the analyzed PAG-sample, which might result from the final step of the purification procedure. The Vicia villosa agglutinin (VVA)-lectin chromatography could selectively bind proteins with multiple Sda-groups and thereby enrich PAGs with a maximal number of Sda-groups on tetraantennary glycans. This posibility was tested by the analysis of N-glycans of the unfractionated proteins of a gd 220 placenta. In this sample, the dominance of the tetraantennary glycan (m/z 5812) over its triantennary counterpart (m/z 4757) is less accentuated than in the PAG-N-glycans, but still visible. This shows that the affinity chromatography probably enriches PAGs which carry tetraanntennary glycans, but that this structure is nevertheless dominating.
The function of bisecting GlcNAc is still not well understood. One interesting finding is that an NK-cell sensitive human cell line (K562) lost its NK-sensitivity after transfection with GlcNAc-TIII, which increases the amount of bisecting GlcNAc on cell surface glycoproteins (Yoshimura et al. 1996). So bisecting GlcNAc seems to reduce target-cell susceptibility for NK-induced cell lysis. Since NK-cells are potentially hazardous for MHC-I-negative trophoblast cells in bovine placentomes (Davies et al. 2000), a local NK-cell directed immunosuppressive function of PAG-glycans could be of great physiological importance. Recently, the ultrastructural localization of BNC-derived PAGs in the maternal placental connective tissue strengthened such speculations about an immunosuppressive function (Wooding et al. 2005).
Several lectin histochemical studies dealt with the glycosylation of BNC secretory granules and demonstrated that GalNAc binding lectins can be used to label these granules with high specificity (Lehmann et al. 1992; Jones et al. 1994; Nakano et al. 2002; Klisch and Leiser 2003). In our present study, we show that the Sda-antigen is the predominant carbohydrate on bovine PAGs in midpregnancy and thereby is the target of the GalNAc-binding lectins. Both, DBA and VVA, recognize the β1,4-linked GalNAc of the Sda-antigen (Wu et al. 1998; Jimenez Blanco et al. 2001), although these lectins are regarded as specific for -linked GalNAc in some publications (Jones et al. 1994; Nakano et al. 2002). The lectin histochemical changes of glycosylation are in accordance with earlier observations, which show that terminal GalNAc is absent in BNC-granules before gd 30 (Lehmann et al. 1992) and at parturition (Klisch et al. 2006). Due to steric hindrance by the ß1,4 linked GalNAc, MAL does not bind to the Sda-antigen (Jimenez Blanco et al. 2001). A simple explanation for the mutually exclusive staining of the BNC with either MAL or CT1 (see Figure 8) would be the absence of ß1,4 linked GalNAc in otherwise unaltered glycans in early pregnancy and at parturition. For parturition, this possibility was ruled out by the analysis of the prepartal N-glycan sample (Figure 3). In this sample, only a few sialylated lactosamine-type glycans were observed. An explanation of the histochemical MAL-staining of BNC at term is difficult, since the mass spectra do not show upregulation of putatively MAL-binding glycans (2,3-sialylated lactosamine) at term. One explanation could be that bulky Sda-glycans mask the MAL-binding sites at midpregnancy, but not before gd 30 and at parturition. Another possibility would be that the highly sensitive MAL-histochemistry detects structures which are represented only by very small peaks in the N-glycan MALDI mass spectra.
In the Western-blot, there is a much more intensive binding of the anti-PAG serum to the glycosylated PAG, compared to the deglycosylated sample. This indicates that the polyclonal serum partially recognizes the attached N-glycans. The Western-analysis also shows that in bovine BNCs, the Sda-antigen is predominantly attached to PAGs and not or to a much lesser extent to another glycoprotein (PRP-I), which is colocalized with PAGs in the BNC granules. This suggests that at least one of the glycosyltransferases, which is involved in the synthesis of the Sda-antigen, recognizes specific features of the protein. This possibility is also supported by the fact that the Sda-antigen has only been demonstrated in a very limited number of glycoproteins, for example, on the N-glycans of Tamm–Horsfall protein (Van Rooijen et al. 1998; Easton et al. 2000a). The Sda-antigen was also found on human and mouse Zona pellucida protein-3 (Easton et al. 2000b) and on CD45 in activated murine cytotoxic T-cells (Lefrancois and Bevan 1985).
The functional relevance of the Sda-antigen remains largely unknown. An involvement in the regulation of PAG serum half-life seems likely. Thereby, the changes of PAG glycosylation in the course of pregnancy may explain changes of serum half-life between the different stages of pregnancy (Klisch et al. 2006). In early pregnancy, the serum half-life of PAGs is approximately 4–5 days (Szenci et al. 2003), while it is around 8–9 days after parturition (Kiracofe et al. 1993). Since the Sda-antigen is a ligand for the asialoglycoprotein receptor in mice (Mohlke et al. 1999), it might accelerate PAG-clearance from the maternal blood. The absence of Sda before gd 30 and at parturition might thereby cause a higher serum half-life of these glycoforms and could thus cause the small peak of PAG-serum levels in the fourth week of pregnancy and also the much more prominent peak at parturition (Green et al. 2005).
We speculated that the absence of the Sda-antigen in the early pregnancy and before parturition might be caused by downregulation of the β4GalNAcT-II transcription at these stages. Therefore, we studied the relative gene expression of this enzyme, but we found no significant differences between the gestational groups. This suggests that the generation of the Sda-antigen is not regulated by the transcription of this enzyme. An alternative explanation would be the regulation at an earlier stage of Sda-synthesis. This could be the downregulation of an 2,3-sialyltransferase, which catalyzes the penultimate step of Sda-synthesis. This would be consistent with the absence of sialylated lactosamine residues in the antepartal MS spectrum (Figure 3).
Pregnancy associated changes of the abundance of the Sda-antigen on red blood cells have been observed in humans (Morton et al. 1970; Spitalnik et al. 1982). The rate of Sda-negative subjects increases with the progression of pregnancy. The Sda-glycotope is absent on erythrocytes of 4% of nonpregnant individuals, while 22% and 36% pregnant women in the first and third trimester are Sda-negative (Spitalnik et al. 1982). In contrast to this, no quantitative changes of the Sda-antigen were observed in the urine of pregnant women (Morton et al. 1970) and there were also no significant changes of the N-glycosylation of Tamm–Horsfall protein (uromodulin) in the course of pregnancy (Van Rooijen et al. 2001). This suggests that the synthesis of the Sda-glycotope is regulated in a tissue-specific way and could be under endocrine regulation. Oestradiol might be the regulatory agent for the changes of PAG-glycosylation in cattle. The BNCs express oestrogen receptor-β (Schuler et al. 2005) and there is a dramatic increase in the concentration of oestrogens in the last week of pregnancy in cattle (Robertson and King 1979). Thus, the rise of oestrogen concentration occurs simultaneously with the disappearance of the Sda-antigen. In early pregnancy, the changes of oestrogen concentrations are less dramatic, but there is a decline of the oestrogen level during the second and third week of pregnancy (Patel et al. 1999). This suggests that the absence of Sda in the gd 20 and 23 samples might be caused by oestrogen. It should also be considered that the local oestrogen concentrations might differ from that in the maternal blood. Thus, it appears that the observed changes of PAG-glycosylation during pregnancy are under endocrine control, but the mechanisms are still unresolved. Our study shows that a singular carbohydrate structure is the predominant constituent of the N-glycans of bovine PAGs. From a glycobiological view, this is very exciting since both the tetraantennary structure in combination with a bisecting GlcNAc and its homogeneity, are highly unusual. In addition, the elucidation of the glycan structure and its changes during pregnancy give new starting points for functional studies which might lead to a better understanding of the function of PAGs.
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Materials and methods |
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Purification of PAGs
PAGs were isolated from cotyledons of one pregnant cow (approximate gestation day (gd) 155) following a recently published protocol (Klisch et al. 2005
). The gd was estimated from the crown-rump length of the fetus (Rexroad et al. 1974). Briefly, the cotyledons were finely minced and homogenized. After ammonium sulfate precipitation (fraction between 40 and 80% saturation) the proteins were dialyzed against Tris-buffer (20 mM Tris-HCl, 2 mM EDTA, pH 7.6) and loaded onto a DEAE-cellulose column. Proteins were eluted with a continuous gradient of increasing NaCl concentrations (0–0.3 M) in Tris-buffer. The fractions were checked for PAG-immunoreactivity by Western-blot with a rabbit polyclonal PAG-antiserum and immunoreactive fractions were pooled. Aliquots of the pooled fractions were incubated with agarose bound Vicia villosa lectin (Vector Laboratories, Burlingame, CA). The column was washed with HEPES-buffer and the proteins were eluted with 25 mM GalNAc in HEPES-buffer. The GalNAc was removed by washing with several volumes of 1 mM Tris-HCl, pH 7.5 on an Amicon Ultra, Centrifugal Filter Device (MWCO 10,000) (Millipore, Schwalbach, Germany) and aliquots of the purified protein were stored at –20°C or lyophilized. The characterization of purified proteins was done by SDS-PAGE and MALDI-MS as published earlier (Klisch et al. 2005) and revealed the same results. Three bands of approximately 56, 66, and 75 kDa were seen in a Coomassie blue-stained gel. These bands were in-gel trypsinated as described (Klisch et al. 2005) and analysis by MALDI-MS identified the proteins as a mixture of PAG-1 (66 kDa), PAG-6 and PAG-7 (75 kDa), and PAG-17 (56 kDa).
MALDI-MS of native PAG-glycans
An aliquot of the purified PAGs (100 µg) was heated (96°C) for 10 min and then the N-glycans were released by digestion with PNGase-F (New England Biolabs, Frankfurt am Main, Germany) in phosphate buffer (pH 7.5) for 24 h at 37°C. The glycans were extracted by graphitised carbon (Alltech Grom, Rottenburg, Germany) after a protocol from Packer et al. (1998), eluted in 75% acetonitrile with 0.15% trifluoroacetic acid and dried in a vacuum centrifuge. An aliquot of the glycans was desialylated with neuraminidase from C. perfringens (Roche, Penzberg, Germany) in 50 mM sodium-acetate buffer, pH 5.0. Samples were analyzed in a MALDI-TOF/TOF mass spectrometer (Ultraflex Bruker Daltonics, Bremen, Germany) at 20 kV in the linear mode. The nondesialylated samples were measured in the negative mode with 20 mg/mL 2',4',6'-trihydroxyacetophenone monohydrate, 20 mM ammonium citrate in 50% acetonitrile as matrix. The desialylated samples were measured in the positive mode with 10 mg/mL 2,5-dihydroxybenzoic acid (DHB) as matrix.
MALDI-MS and MS/MS analysis of permethylated PAG N-glycans
A sample of approximately 50 µg of the purified PAG was reduced for 1 h at 37°C in 50 mM Tris-HCl buffer (pH 8.5) containing a fourfold excess of dithiothreitol and carboxymethylated with a twofold molar excess of iodoacetic acid for 1 h at room temperature in the dark. Following dialysis at 4°C for 72 h against 4 x 4.5 litres of cold 50 mM ammonium bicarbonate, pH 7.5, and lyophilization, the sample was digested with sequencing-grade trypsin (Promega, Mannheim, Germany) (1 µg in 50 mM ammonium bicarbonate, pH 8.5, for 18 h at 37°C). The reaction was stopped by adding a few drops of acetic acid to the solution. The sample was lyophilized prior to its dissolution in 150 µL (5% (v/v)) acetic acid and purified using a Sep-Pak cartridge C18 (Waters Corp, Eschborn, Germany), as previously described (Jang-Lee et al. 2006). The purified glycopeptides were digested with PNGase-F (Roche Applied Science) in 50 mM ammonium bicarbonate (pH 8.5) containing 10 units of enzyme at 37°C over 18 h. The sample was lyophilized, and the released N-glycans were purified using a Sep-Pak cartridge C18 (Waters Corp). Permethylation and sample cleanup were performed using the sodium hydroxide protocol, as described previously (Jang-Lee et al. 2006). Preparation of partially methylated alditol acetates was performed as described (Jang-Lee et al. 2006).
MALDI-TOF MS data on permethylated samples were acquired in positive ion mode (M+Na)+ using a Perseptive Biosystems Voyager DE-STRTM mass spectrometer in the reflector mode with delayed extraction. MS/MS data were acquired using a 4800 MALDI-TOF/TOF (Applied Biosystems, Foster City, CA) mass spectrometer. The collision energy was set to 1 kV, and argon was used as collision gas. Samples were dissolved in 10 µL of methanol and 1 µL was mixed at a 1:1 ratio (v/v) with DHB as matrix.
Glycomics analysis of placental tissues
Placental tissues (1 g each) from two pregnant cows (gd 220 and one day before birth) were homogenized in six volumes of homogenization buffer (10 mM HEPES, 150 mM NaCl, pH 7.5) and subsequently lyophilized. For N-glycan analysis, approximately 80 mg of the homogenized placental tissues were subjected to the same procedures as for the permethylated PAG N-glycans. For O-glycan analysis, the same amount of homogenized placental tissues was subjected to reductive elimination by adding 400 mL of 1 M potassium borohydride (54 mg/mL in 0.1 M potassium hydroxide) for 24 h incubation at 45°C. The reaction was terminated by a dropwise addition of glacial acetic acid followed by Dowex chromatography and borate removal using 10% of methanolic acetic acid (Jang-Lee et al. 2006). The purified O-glycans were then permethylated and the mass spectrometric analyses were carried out as described in the earlier section for permethylated PAG N-glycans.
Western analysis
Proteins from homogenized placentomal tissue of a late pregnant cow (approximately gd 260) were either deglycosylated with PNGase F or left nondeglycosylated. The samples (10 µg/lane) were separated by SDS-PAGE and transferred to a PVDF membrane as described earlier (Klisch et al. 2005). The membrane was probed with CT1-antibody (1:500, concentrated cell-culture medium of the CT1-hybridoma, gift from L Lefrancois, University of Connecticut, Farmington, CT), anti-PAG (1:10,000; rabbit antiserum R727, gift from JF Beckers, University Liege, Liege, Belgium), and PRP-I (1:20,000; gift from L Schuler, University of Wisconsin-Madison, Madison, WI). Secondary antibodies were peroxidase-labeled anti-IgM mouse (Sigma, Deisenhofen, Germany) and anti rabbit (GE-Healthcare, Freiburg, Germany). Blots were developed with enhanced chemiluminescence (ECL Plus Western Blotting Detection Reagents, GE-Healthcare) after washing in PBS.
Immuno- and lectin histochemistry
Uteri of early pregnant cows (gd 20, 23, 32, 37; each n = 1) were fixed by a perfusion with 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.4). Pieces of the chorionic sac were removed from the uteri and further fixed by immersion in 3% glutaraldehyde and embedded in epon as described (Klisch and Leiser 2003). Bovine placentomal tissues of mid pregnancies (gd 94, 110, 154, 198) were collected at a slaughterhouse. Tissues of preterm (caesarean sections either 27 h after a Prostaglandin F2-alpha analogon injection approximately 5 days (approximately gd 275) before the expected end of pregnancy (n = 3) or after the prepartal decline of maternal progesterone became obvious (approximately 1 day ante partum; n = 2) and term placentomes (approximately gd 280; n = 3) were obtained by caesarean sections as described earlier (Klisch et al. 2006; Schuler et al. 2006). The tissues were fixed in 4% formaldehyde (v/v) in 0.1 M phosphate buffer (pH 7.3) for 24 h and embedded in paraffin.
Paraffin sections (7 µm) were dewaxed in xylol, rinsed in three changes of ethanol, rehydrated in descending concentrations of ethanol, and rinsed in distilled water. Epon sections (0.5 µm) were deplasticized and rehydrated as described earlier (Klisch and Leiser 2003). The slides were rinsed in 0.05 M TRIS-buffered saline, pH 7.6, 1 mM CaCl2 (TBS), and incubated for 45 min in a humid chamber at 37°C with 10 µg/mL biotinylated lectin (DBA, Sigma; PHA-L, EY-Laboratories, San Mateo, CA; MAL-I, Vector Laboratories) in TBS. For double stainings, the sections were incubated with the carbohydrate-binding reagent (biotinylated lectins or CT1-antibody) in combination with anti-PAG. Lectins were visualized with streptavidin-Cy3, CT1 by Cy3 anti-mouse, and the polyclonal anti-PAG by Cy2-anti-rabbit. In controls, the lectin or primary antibody were replaced by buffer. In additional controls, 0.2 M GalNAc was added to the buffer during incubation with DBA. As a control for the CT1-antibody, an irrelevant mouse IgM antibody (monoclonal antibody to single-stranded DNA [F7–26]; Alexis Biochemicals, Gruenberg, Germany) was used instead of the CT1-antibody. The BNCs were identified by the PAG-immunostaining and in the doublestainings, the fractions of MAL-, DBA-, PHA-L- and CT1-positive BNC were evaluated. For each lectin, and also for the CT1 antibody, approximately 30 fields of vision (each 0.173 mm2) were evaluated for each animal. Due to the small size of the sections of the earliest stages (gd 20; gd 23), all BNCs were evaluated in these specimens.
Quantitative PCR of β4GalNAcT-II
Based on a predicted sequence of the bovine β-4-N-acetylgalactosaminyltransferase (β4GalNAcT-II) mRNA (GenBank Accession Number XM_584835
[GenBank]
), which has 75% and 74% amino acid homology to the human and mouse β4GalNAcT-II-mRNA respectively, we confirmed the existence of the corresponding transcript in bovine placentomes by PCR. We amplified a 847 bp fragment (forward primer 5'-AGG GTG GAT GTG GTG AGT CT-3'; reverse primer: 5'-CAC ATT GGA GGT GGT TCT TG-3') on bovine placentomal cDNA. This fragment was sequenced and submitted to genbank (GenBank Accession Number EF445547
[GenBank]
). This sequence was used for the quantification of the mRNA with the TaqMan system (forward primer 5'-GTG GCT GAT GAC AGC AAG GA-3': reverse primer 5'-GCC GTA GGG CAT GGT GTA AT-3'; TaqMan Probe: 5'-CCC CTG AAA ATT AAT GAC AGC CAT GTG G-3') as described (Kowalewski et al. 2006). Relative mRNA levels for bovine β4GalNAcT–II and for the housekeeping gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) were determined in one placentome of each of the 14 cows assigned to four observational groups, representing the midgestation (day 100–200; n = 3), late gestation (day 200–280; n = 5), the prepartal decline in maternal progesterone concentrations (approximately one day before the normal onset of parturition, n = 3) and parturition (n = 3) (Schuler et al. 2006).
Primer Express software (version 2.0, Applied Biosystems) was used to design primers and TaqMan Probe (forward primer 5'-GTG GCT GAT GAC AGC AAG GA-3': reverse primer 5'-GCC GTA GGG CAT GGT GTA AT-3'; TaqMan Probe: 5'-CCC CTG AAA ATT AAT GAC AGC CAT GTG G-3'). The primers were ordered from MWG Biotech AG, Ebersberg, Germany, the TaqMan probe was from Eurogentec, Seraing, Belgium. TaqMan probe was labeled at the 5'- end with reporter dye 6-carboxyfluorescein (FAM) and at the 3'- end with the quencher dye 6-carboxytetramethyl-rhodamine (TAMRA).
ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Darmstadt, Germany) was used and experiments were performed according to our previously described protocol (Kowalewski et al. 2006). Briefly: 200 ng of total RNA was DNase-treated and reverse-transcribed as in the routine RT-PCR. Samples were analyzed in duplicates; 25 µL of reaction mixture contained 12.5 µL TaqMan® qPCR MasterMix (Eurogentec), 300 nM of each primer and 200 nM TaqMan Probe, and 5 µL of cDNA. Amplification was carried out as follows: denaturation for 10 min at 95°C followed by 40 cycles at 95°C for 15 s and 60°C for 60 s.
Relative quantification was done by normalizing the β4GalNAcT-II signals with the GAPDH signal (as "housekeeping gene") using the comparative CT method (
CT method) according to the instructions of the manufacturer of the ABI PRISMTM 7000 Sequence Detector. The threshold cycle (CT) represents the PCR cycle at which an increase in reporter fluorescence above a base line signal can first be detected.
Finally, relative gene expression (RGE) was calculated as expression of the target gene relative to the reference gene (GAPDH) and normalized to the calibrator (sample with the lowest amounts of the respective target gene transcripts). Briefly, the analysis was performed as follows: For each mRNA, a difference in CT values (CT) was calculated by taking the mean CT of duplicate tubes and subtracting the mean CT of the duplicate tubes for the reference RNA (GAPDH) measured in an aliquot from the same RT reaction (CT = CT test gene – CT GAPDH; treated sample). The CT for the treated sample was then subtracted from the CT for the calibrator to generate a CT (CT = CT treated sample – CT calibrator). The mean of these CT measurements was then used to calculate expression of the test gene (2-CT) relative to the reference gene (GAPDH) and normalized to the calibrator (relative gene expression (RGE) = 2–CT).
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Deutsche Forschungsgemeinschaft (DFG) (KL 1835/1-1 to K.K.); Biotechnology and Biological Sciences Research Council; Wellcome Trust (to A.D.).
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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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The authors would like to thank Ms Silke Fischer for excellent laboratory assistance. The PAG-antiserum was a gift from J.-F. Beckers, University of Liege, Liege, Belgium and the PRP-I antiserum was obtained from L. Schuler, University of Wisconsin-Madison, Madison, USA. The CT1-hybridoma was a gift from L. Lefrancois, University of Connecticut, Farmington, CT, USA. P.-C.P. is a recipient of Imperial College London studentships and A.D. is a Biotechnology and Biological Sciences Research Council (BBSRC) Professorial Fellow.
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Footnotes |
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1Present address: School of Veterinary Medicine and Science, University of Nottingham, Loughborough LE11 5RD, UK
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Abbreviations |
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BNCs, binucleate trophoblast giant cells; d a.p., days ante partum; DBA, Dolichos biflorus agglutinin; DHB, 2,5-dihydroxy-benzoic acid; GalNAc-T, N-acetylgalactosaminyltransferase; GAPDH, glycerinaldehyd-3-phosphat-dehydrogenase; gd, gestation day; GlcNAc, N-acetylglucosamine; GlcNAc-T, N-acetylglucosaminyltransferase; MAL, Maackia amurensis lectin; MALDI-MS, matrix assisted laser desorption ionisation-mass spectrometry; Man, mannose; MW, molecular weight; NeuAc, N-acetylneuraminic acid; PAG, pregnancy-associated glycoprotein; PHA-L, Phaseolus vulgaris leucoagglutinin; PRP-I, prolactin related protein-I; TOF, time of flight; Sda, NeuAc
2–3[GalNAcβ1–4]Galβ1–4GlcNAc; VVA, Vicia villosa agglutinin |
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