Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo.

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.

The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse.

BACKGROUND: In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, since the Galactosyltransferase and N-Acetylglucosamine residues have been localized on equine gametes. METHODS: We masked the Galactosyltransferase and N-Acetylglucosamine residues before the co-incubation of gametes. Galactosyltransferase was masked either with an anti-Galactosyltransferase antibody or with the enzyme substrate, UDP Galactose. N-Acetylglucosamine residues were masked either with a purified Galactosyltransferase or with an anti-ZPC antibody. RESULTS AND DISCUSSION: The number of spermatozoa bound to the zona pellucida did not decrease after the masking of Galactosyltransferase or N-Acetylglucosamine. So, these two molecules may not be necessary in the mechanism of in vitro sperm-zona pellucida interaction in the horse. CONCLUSION: The involvement of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding may have been lost during evolution in some ungulates, such as porcine and equine species.

Differentially expressed long non­coding RNAs and mRNAs in patients with IgA nephropathy.

Long non­coding RNAs (lncRNAs) have been reported to serve a crucial role in renal diseases; however, their role in immunoglobulin A nephropathy (IgAN) remains unclear. In the present study, peripheral blood mononuclear cells (PBMCs) were collected from both patients with IgAN and healthy controls. A microarray analysis was then performed to identify differentially expressed lncRNAs and mRNAs in PBMCs, which were confirmed by quantitative polymerase chain reaction. In addition, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and lncRNA­mRNA co­expression network analyses were conducted. The present study identified 167 differentially expressed lncRNAs and 94 differentially expressed mRNAs. Numerous GO terms, including innate immune response, inflammatory response, IPAF inflammasome complex and UDP­galactose:ß­N­acetylglucosamine ß­1, and 3­galactosyltransferase activity, were significantly enriched in the differentially expressed mRNAs. The top five KEGG signaling pathways included nucleotide­binding oligomerization domain­like receptor signaling pathway, hematopoietic cell lineage, inflammatory bowel disease, tumor necrosis factor signaling pathway and other types of O­glycan biosynthesis. In addition, a total of 149 lncRNAs were shown to interact with 7 mRNAs that were associated with the 'innate immune response' GO term. The results of the present study demonstrated that differentially expressed lncRNAs and mRNAs may have a role in the development of IgAN. These results may aid in the elucidation of a basic pathogenic mechanism, the identification of possible biomarkers and the generation of potential novel treatment strategies for IgAN.

Modification of the cytoplasmic domain affects the subcellular localization of Golgi glycosyl-transferases.

Our goal was to engineer a Golgi glycosyltransferase epitope-tagged on its cytoplasmically exposed, short, N-terminal domain that gave normal subcellular localization. Partial replacement of the cytoplasmic tail of human alpha-2,6-sialyltransferase (SialylT) with the negatively charged myc or FLAG epitope resulted in almost complete mislocalization of the chimera expressed in Vero cells. A granular cytoplasmic staining pattern was seen by immunofluorescence. Spacing the negatively charged residues progressively outward from the negative N-terminus resulted in increasingly more normal localization of myc or FLAG-tagged protein to a juxtanuclear Golgi-like distribution. Substitution of a neutrally charged VSV-G sequence for these tags resulted in normal localization of the chimera to the juxtanuclear Golgi region. Insertion of the myc epitope within the N-terminal domain of the short form of bovine beta-1,4-galactosyltransferase (GalT) gave a chimeric protein that mislocalized in BHK cells. No signal was detected with a monoclonal anti-epitope antibody indicating that the myc epitope was masked. Placement of myc or FLAG epitopes at the NH2-terminus of human N-acetylglucosaminyltransferase I (GlcNAc-T) resulted in chimeric proteins that in Vero cells displayed little Golgi localization. We conclude that positioning of negative charge, in particular, close to the membrane, typically produces a failure of type II Golgi glycosyltransferases to exit the ER/CGN, presumably due to quality control mechanisms. These proteins may be successfully epitope-tagged on their N-terminal domain either using a neutral or positively charged sequence or spacing any negatively charged sequence out from the membrane.

Distinct compartmentalization of TGN46 and beta 1,4-galactosyltransferase in HeLa cells.

The Golgi proteins, TGN46 and GalT, were characterized in human HeLa cells using specific polyclonal and monoclonal antibodies. A bacterially expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN46 had an apparent molecular mass of 100 to 120 kDa which reflects extensive glycosylation. Epifluorescence light microscopy indicated substantial colocalization of TGN46 and GalT. However, confocal laser microscopy and three-dimensional reconstruction of double-labeled HeLa cells revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approximately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was detected in tubules. Distinct GalT-positive regions within the Golgi cisternal stack were not labeled for TGN46.

A protein-specific monoclonal antibody to rat liver beta 1-->4 galactosyltransferase and its application to immunohistochemistry.

We have produced a new protein-specific monoclonal antibody (MAb) to rat liver beta 1-->4 galactosyltransferase. This MAb, GTL2, was selected as the most reactive IgG to a periodate-treated antigen. Antigen and protein specificities of GTL2 were verified by immunoblotting of a non-glycosylated recombinant protein of human galactosyltransferase and enzymatically deglycosylated rat galactosyltransferase. Using GTL2, an immunohistochemical study was done in rat liver, epididymis, and salivary glands. Intense staining was observed in Golgi areas of epididymal duct epithelial cells, and submandibular and sublingual acinar cells. Hepatocytes showed weaker staining. Immunoelectron microscopic observation revealed that the staining was exclusively localized in trans-Golgi membranes of these cells.

Antibody recognition of epitopes on wild-type and mutant beta-(1-->4)-galactosyltransferase-1.

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.

Purification and characterization of recombinant human beta 1-4 galactosyltransferase expressed in Saccharomyces cerevisiae.

A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell beta-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum. Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly. This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.

Immunocytochemical localization of the protein reactive to human beta-1, 4-galactosyltransferase antibodies during chick embryonic skin differentiation.

BACKGROUND: beta-1, 4-Galactosyltransferase (GalTase) transfers galactose from UDP-galactose to terminal N-acetylglucosamine in glycoconjugates and is located both in the Golgi apparatus and in the plasma membrane. The cell surface GalTase is thought to be involved in cell-to-cell recognition and cell-to-extracellular matrix interaction. METHODS: By the use of specific monoclonal antibodies against human GalTase, changes in cell surface localization of the protein reactive to the antibodies in chick embryonic skin during its differentiation in vivo and in vitro were detected immunohistochemically at both light- and electron microscopic levels. The distribution of glycoconjugates having terminal N-acetylglucosamine residues was detected by staining with succinylated wheat germ agglutinin (s-WGA). RESULTS: Under the light microscope, intense immunostaining was observed in the keratinized epidermis, particularly in the intermediate layer. Marked changes in the localization of the staining were observed in vitamin A-induced mucus-secreting skin, in which keratinization was suppressed. The localization of the immunostaining was in parallel with that of glycoconjugates having terminal N-acetylglucosamine residues. Immunoelectron microscopically the immunostaining was located on the cell surface and in the intercellular space of the desmosomes in the intermediate cells of the keratinized epidermis. However, the staining was not present on the cell surface but was detected on the limiting membrane of the mucous granules, in the mucous metaplastic epidermis. In contrast, the staining was always found in the Golgi apparatus in all of the cells. CONCLUSIONS: These results suggest that the protein reactive to human GalTase antibody may be involved in chick epidermal differentiation.

Effect of peptide acetylation on its interaction with antipeptide antibody.

The effects of trace acetylation on the contact sites in peptide-antibody binding have been investigated by using a label selection method. A 13-residue synthetic peptide having three lysines (LKLVEQQNPKVKL) corresponding to sequence position 155-168 of beta 1,4-galactosyltransferase was used in this study. The four amino groups located throughout the peptide sequence, labeled appropriately, served as probing marker groups. The amino terminus region of the peptide was highly reactive to trace acetylation. Over 80% of the label appeared in the amino terminus region, most likely in the alpha-amino group due to its lower pK value. Interestingly, acetylation of Lys10 and Lys12 (carboxy terminal region) showed a selection for interacting with the antibody. This approach, using label selection affords high sensitivity and could be applicable for mapping antigen-antibody interaction site/s.

Cell surface galactosyltransferase: immunochemical localization.

A cell surface UDP-galactose:N-acetylglucosamine galactosyltransferase (GT) has been directly localized on bovine cells in tissue culture by immunohistochemical techniques. A conventional rabbit heteroantiserum was prepared against an affinity-purified soluble form of GT from bovine milk, and a monospecific IgG fraction was isolated by affinity chromatography on a GT adsorbent. As demonstrated by indirect immunofluorescence, antigen to this antibody is present on the surface of all three bovine cell lines tested. It was uniformly distributed over the exposed membrane surface of fixed cells. Exposure of living cells to the anti-GT antibody resulted in its time-dependent aggregation in the plane of the membrane. Antigen (GT) was released from the membrane surface by trypsin digestion, and its reappearance required protein synthesis, since cycloheximide effectively prevented repopulation of the cell surface.

Production of recombinant xenotransplantation antigen in Escherichia coli.

The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug. As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection. An E. coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA. This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.