Notch (N) is a transmembrane receptor that mediates cell–cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP--fucose as a donor of fucose. The GDP--fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy--mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP--fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP--fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP--fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP--fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP--fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.
In our study, we analyze the alternations in the expression of MAA- and SNA-reactive glycotopes and LCA-, LTA-, and UEA-reactive glycotopes of IgA in amniotic fluid during the normal human pregnancy from the 2nd trimester, throughout the 3rd trimester, perinatal period, post-date pregnancy and delivery. The levels of sialylation and fucosylation of the IgA were determined by lectin-IgA-ELISA using lectins with known specificity toward different types of sialic acid and fucose linkages Table [–]. The sialylation and fucosylation patterns were determined on plasma and amniotic IgA, isolated by modified polyclonal anti-α chain antibodies. It should be pointed out that this approach did not determine the ‘true’ structure of the human IgA glycans, but allowed to observe the changes of glycotope expressions accessible to the interactions with lectins. Such glycan-lectin interactions between the lectin-receptors and the sialyl- and fucosyl-glycotopes are emerging as key elements in a wide range of pathophysiological processes.
Fucose modifications, including protein O-fucosylation, require GDP--fucose as the fucose donor. In mammals, GDP--fucose is synthesized by both de novo and salvage pathways (). However, in Drosophila, GDP--fucose is synthesized only by the de novo pathway (, ). Thus, Drosophila is a useful system for studying the requirement of GDP--fucose in various biological events (). The enzymes essential for the de novo synthesis of GDP--fucose, GDP-mannose 4,6 dehydratase (Gmd), and GDP-4-keto-6-deoxy--mannose 3,5-epimerase/4-reductase (Gmer), are encoded by the Gmd and Gmer genes, respectively (). The Gmd homozygote causes GDP--fucose starvation in Drosophila ().
In general, the biosynthetic cascades of nucleotide sugars, including GDP--fucose, are considered intracellular events, although sugars can also be acquired from the extracellular space through specific transporters (). However, a previous study demonstrated that UDP-galactose and UDP-GalNAc are transported intercellularly among cultured CHO cells through intercellular junctions (). A CHO cell derivative that is deficient in the enzyme UDP-Gal/UDP-GalNAc 4-epimerase shows defective LDL receptor structure and activity, which are restored by cocultivation with cells expressing the normal activity of this enzyme (). This restoration of LDL receptor activity is suppressed by adding retinoic acid, an inhibitor of junctional communication (). These results suggest that UDP-galactose and UDP-GalNAc can be supplied through intercellular junctional communication. However, such intercellular transport of nucleotide sugars has not been demonstrated in vivo. Furthermore, the nature of the intercellular communication involved in this delivery of nucleotide sugars is not yet clear. To address these issues, here we studied the intercellular delivery of GDP--fucose into cells in vivo, using mutants of genes involved in the biosynthetic pathway of GDP--fucose in Drosophila.
Although the enzymes required for the biosyntheses of various nucleotide sugars have been elucidated (), very few studies have examined whether nucleotide sugars are exchanged intercellularly (), and none have investigated such exchanges in vivo. To address these issues, we next examined whether Gmd and Gmer function cell-autonomously or cell-nonautonomously in vivo, because these mutants should behave cell-nonautonomously if GDP--fucose is transferred intercellularly. Although the cell-nonautonomous behavior of Gmd was previously reported (, , ), this phenomenon has not been examined in detail.
The nucleotide sugars UDP-galactose and UDP-GalNAc were previously shown to be transported although intercellular junctions in mammalian cultured cells, although the involvement of intercellular junctional communication was based only on an inhibitor with low specificity, which was available at that time (). In this study, we demonstrated that GDP--fucose is intercellularly exchanged among epithelial cells within an organ through inx2-dependent gap junctions in vivo. Thus, this is evidence for the intercellular exchange of a nucleotide sugar through gap junctions in a living animal. This intercellular supply of GDP--fucose is sufficient for the O-fucosylation of N, even in the absence of its cell-autonomous supply by de novo biosynthesis.
First, we overexpressed Gmd or Gmer in its respective homozygous mutant along the anterior–posterior compartment boundary (A/P boundary) of wing imaginal discs (GmdH78 and GmerSH; ). The overexpressed constructs were driven by decapentaplegic (dpp)-Gal4 in the region indicated by the expression of GFP (). Despite the restricted expression of these genes in the wing imaginal discs, the expression of Wg was rescued along the entire D/V boundary in all cases examined (n > 20) (). We also found that the specific expression of Gmd driven by breathless (btl)-Gal4 in the trachea blast, which is attached to the narrow portion of the wing imaginal disc (, GFP-positive region indicated by arrow), was sufficient to rescue the Wg expression along the entire wing imaginal disc D/V boundary in GmdH78 homozygotes, in all cases examined (n > 20) (). The AAL staining of these wing imaginal discs was also restored (). These results indicate that GDP--fucose can spread throughout the epithelium of the wing imaginal discs.
In our opinion, the pregnancy-associated unique expressions of sialylated and fucosylated glycoforms of amniotic IgA illustrate a general importance of carbohydrate-lectin and/or carbohydrate-carbohydrate interactions in the control and modulation of pro- and anti-inflammatory events to ensuring the fetus well-being. Glycovariants of amniotic IgA associated with pregnancy progression overlap and/or are influenced by a gestational-depended pro- and anti-inflammatory condition. They seem to be molecular indicators reflecting the pregnancy age and can also be used as a window into the physiological events that lead up to both normal and post-date pregnancy. However, a future aim for these studies is the structural analysis of the IgA oligosaccharides that should confirm or verify relative levels of sialic acid and fucose of the lectin based proposals, and helps to understand their role and impact on the course of pregnancy.