In bovine binucleate trophoblast giant cells,pregnancy-associated glycoproteins and placental prolactin-related protein-I are conjugated to asparagine-linked<Emphasis Type="Italic"> N</Emphasis>-acetylgalactosaminyl glycans

Bovine binucleate trophoblast giant cells (BNCs) produce large amounts of PAS-positive cytoplasmic granules. After fusion of BNCs with uterine epithelial cells, the contents of these granules are released into the maternal stroma which underlies the uterine epithelium. Histochemically, the granules can be labeled with N-acetylgalactosamine-specific lectins ( Dolichos biflorus, Vicia villosa, and Wisteria floribunda agglutinins) and with Phaseolus vulgaris leucoagglutinin. In this study, we used lectin western blot analysis of proteins from fetal cotyledons to characterize the lectin binding glycoproteins. Lectin western blots showed several bands. A main band of approximately 65 kDa was identified as pregnancy-associated glycoproteins (PAGs) and a double band at 34-35 kDa as prolactin-related protein-I (PRP-I) by their crossreactivity with specific antisera. Enzymatic cleavage of N-linked glycans with peptide- N-glycanase F abolished the lectin binding to PRP and PAGs in western blots, revealing that the lectins bound to asparagine-linked glycans. The high specificity of the lectins was used for the enrichment of PRP-I and PAGs from placental cotyledons with Vicia villosa lectin affinity chromatography. The occurrence of the relatively uncommon asparagine-linked N-acetylgalactosaminyl glycans on secretory proteins of the BNCs suggests a functional role of this specific glycosylation pattern.     

The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers

The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80-100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a 'sandwich' type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (approximately 70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 +/- 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 +/- 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.     

Control of binucleate cell migration in the placenta of sheep and goats

In the ruminant placenta 15-20% of the trophectodermal epithelium consists of granulated binucleate cells (BNC). In the sheep the granules contain ovine placental lactogen (oPL). These cells migrate from the trophectoderm to form fetomaternal hybrid tissue from implantation to term. The number of BNC, their percentage migration and the potential secretory activity of the syncytium they form were estimated by semiquantitative transmission electron-microscopical techniques after several surgical techniques and hormone or drug infusions. BNC numbers decrease normally just before parturition, and this fall could be eliminated by fetal hypophysectomy or induced early by administration of tetracosactrin to intact or hypophysectomized fetuses. If only one twin was treated with tetracosactrin the placenta of the untreated twin did not show the fall in BNC numbers found in the other unless it died in utero some time before sampling. This indicates fetal control of BNC number and migration. However, fetal catheterization, hypophysectomy, stalk section, adrenalectomy, infusion of mouse epidermal growth factor or bromocriptine had little or no effect on binucleate cell numbers or migration percentages. Maternal carunclectomy, ovariectomy, or epostane or bromocriptine administration also had no consistent significant effect. Previous reports of degeneration of BNC structure plus a decrease in their number (with bromocriptine) or an increase in migration frequency (after adrenalectomy or stalk section) have not been confirmed by this study. The BNC migration delivers the oPL-containing BNC granules close to the maternal circulation but the variation in migration seems only loosely correlated with the reported maternal oPL concentrations. The results indicate that BNC migration is independent of the hormonal milieu, but that BNC production is greatly modified by the hormonal changes just before parturition, with cortisol production by the fetus a possible primary cause.     

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

Autoimmunization of ewes against pregnancy-associated glycoproteins does not interfere with the establishment and maintenance of pregnancy

Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n = 22) or with KLH alone (n = 9), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.6 ± 2.2 of pregnancy. Those ewes immunized against PAGs (n = 7), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.9 ± 1.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n = 15), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1 ± 15.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.     

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.     

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.     

beta1,4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells

Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis.     

Characterization of the N-linked oligosaccharides of megalin (gp330) from rat kidney

Megalin (gp 330) is a large cell surface receptor expressed on the apical surfaces of epithelial tissues, that mediates the binding and internalization of a number of structurally and functionally distinct ligands. In this paper we report the first detailed structural characterization of megalin-derived oligosaccharides. Using strategies based on mass spectrometric analysis, we have defined the structures of the N-glycans of megalin. The results reveal that megalin glycoprotein is heterogeneously glycosylated. The major N-glycans identified belong to the following two classes: high mannose structures and complex type structures, with complex structures being more abundant than high mannose structures. The major nonreducing epitopes in the complex-type glycans are: GlcNAc, Galbeta1-4GlcNAc (LacNAc), NeuAcalpha2-6Galbeta1-4GlcNAc (sialylated LacNAc), GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc (Sd(a)) and Galalpha1-3Galbeta1-4GlcNAc. Most complex structures are characterized by the presence of (alpha1,6)-core fucosylation and the presence of a bisecting GlcNAc residue.     

A cloning and expression analysis of pregnancy-associated glycoproteins expressed in trophoblasts of the white-tail deer placenta

The pregnancy-associated glycoproteins (PAGs) are placental proteins that have been cloned from swine, sheep, goats, and cattle, but never from animals within the Cervidae family. The goal of this work was to characterize PAGs in white-tailed deer. Placenta and uterine tissues were collected from pregnant does at days 85 and 90 of pregnancy. RNA from cotyledons was used to amplify deer PAGs by RT-PCR. Ten distinct cDNAs were cloned and sequenced. Some normally conserved amino acids comprising the catalytic site were found to be altered in deer PAGs 4, 5, and 8; another PAG, (PAG-9) was a splice variant that lacked exon 7. In each case, these mutations would likely preclude proteolytic activity for these proteins. A phylogenetic analysis revealed that most of the deer PAGs fell within the ancient PAG grouping. The remainder fell within the more modern (BNC-specific) PAG group. Western blotting was performed with anti-PAG antibodies and this analysis revealed that deer PAGs comprise a heterogeneous group based on different antigenicities and electrophoretic mobilities. Immunohistochemistry and in situ hybridization revealed some unique localization patterns of PAGs in the deer placentome compared to those in other ruminants. Most notably, deer PAGs 4 and 5, which according to the phylogeny, are "ancient PAGs," were expected to be present in all trophoblasts; instead, they were localized to the BNC. Although many of the PAGs identified here are very similar to those in Bovidae, some are clearly distinct in their expression pattern and probably possess functional roles unique to cervid reproduction.     

The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern

Abstract Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-ēpimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells pres\xadented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

Donor specificity in the glycosylation of Tamm-Horsfall glycoprotein: conservation of the Sda determinant in pairs of twins

The content of the Sd(a) determinant in urinary human Tamm-Horsfall glycoprotein (THp) has been reported to be donor-specific. This feature was further addressed by investigating THp from genetically identical individuals. To this end, THp was isolated from the urine of two monozygotic pairs of twins (A and B). The four samples (THp A1, A2, B1, and B2) were subjected to endo-beta-galactosidase from Bacteroides fragilis leading to the liberation of the Neu5Ac(alpha2-3)Gal (beta1-4)GlcNAc(beta1-3)Gal and Neu5Ac(alpha2-3)[GalNAc(beta1-4)] Gal(beta1-4)GlcNAc(beta1-3)Gal (Sd(a) epitope) motifs, both located at the nonreducing termini of complex type N-glycans. The isolated mixtures of oligosaccharides were analyzed for the absolute and relative amounts of the two oligosaccharides. The obtained data clearly indicate that in THp A1 and A2, and in THp B1 and B2, the molar ratios of the tetra- and Sd(a) pentasaccharide are identical for a pair of twins. This conservation of molar ratios points to an identical relative expression of beta-1,4-N-acetylgalactosaminyltransferase activity involved in the biosynthesis of the Sd(a) determinant. Apparently, the degree of conversion of the tetrasaccharidic Sd(a) precursor into the final pentasaccharidic Sd(a) form can be considered to result from a very closely related pattern of glycosylation for genetically homogeneous individuals.     

Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin

beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.     

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.     

Immunogold co-localization of ovine placental lactogen and the antigen recognized by the SBU-3 monoclonal antibody in sheep placental granules

Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function. In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.     

The bisecting GlcNAc in cell growth control and tumor progression

The bisecting GlcNAc is transferred to the core mannose residue of complex or hybrid N-glycans on glycoproteins by the β1,4-N-acetylglucosaminyltransferase III (GlcNAcT-III) or MGAT3. The addition of the bisecting GlcNAc confers unique lectin recognition properties to N-glycans. Thus, LEC10 gain-of-function Chinese hamster ovary (CHO) cells selected for the acquisition of ricin resistance, carry N-glycans with a bisecting GlcNAc, which enhances the binding of the erythroagglutinin E-PHA, but reduces the binding of ricin and galectins-1, -3 and -8. The altered interaction with galactose-binding lectins suggests that the bisecting GlcNAc affects N-glycan conformation. LEC10 mutants expressing polyoma middle T antigen (PyMT) exhibit reduced growth factor signaling. Furthermore, PyMT-induced mammary tumors lacking MGAT3, progress more rapidly than tumors with the bisecting GlcNAc on N-glycans of cell surface glycoproteins. In recent years, evidence for a new paradigm of cell growth control has emerged involving regulation of cell surface residency of growth factor and cytokine receptors via interactions and cross-linking of their branched N-glycans with a lattice of galectin(s). Specific cross-linking of glycoprotein receptors in the lattice regulates their endocytosis, leading to effects on growth factor-induced signaling. This review will describe evidence that the bisecting GlcNAc of N-glycans regulates cellular signaling and tumor progression, apparently through modulating N-glycan/galectin interactions.