The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.     

Glycoengineering of alphaGal xenoantigen on recombinant peptide bearing the J28 pancreatic oncofetal glycotope

In human pancreatic adenocarcinoma, alterations of glycosylation processes leads to the expression of tumor-associated carbohydrate antigens, representing potential targets for cancer immunotherapy. Among these pancreatic tumor-associated carbohydrate antigens, the J28 glycotope located within the O-glycosylated mucin-like C-terminal domain of the fetoacinar pancreatic protein (FAPP) and expressed at the surface of human tumoral tissues, can be a good target for anticancer therapeutic vaccines. However, the oncodevelopmental self character of the J28 glycotope associated with the low immunogenicity of tumor-associated carbohydrate antigens may be a major obstacle to effective anti-tumor vaccine therapy. In this study, we have investigated a method to increase the immunogenicity of the recombinant pancreatic oncofetal J28 glycotope by glycoengineering Galalpha1,3Galss1,4GlcNAc-R (alphaGal epitope) which may be recognized by natural anti-alphaGal antibody present in humans. For this purpose, we have developed a stable Chinese hamster ovary cell clone expressing the alphaGal epitope by transfecting the cDNA encoding the alpha1,3galactosyltransferase. These cells have been previously equipped to produce the recombinant O-glycosylated C-terminal domain of FAPP carrying the J28 glycotope. As a consequence, the C-terminal domain of FAPP produced by these cells carries the alphaGal epitope on oligosaccharide structures associated with the J28 glycotope. Furthermore, we show that this recombinant "alpha1,3galactosyl and J28 glycotope" may not only be targeted by human natural anti-alphaGal antibodies but also by the mAbJ28, suggesting that the J28 glycotope remains accessible to the immune system as vaccinating agent. This approach may be used for many identified tumor-associated carbohydrate antigens which can be glycoengineered to carry a alphaGal epitope to increase their immunogenicity and to develop therapeutic vaccines.     

Anti-alpha-galactosyl antibodies recognizing epitopes terminating with alpha1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.     

Specificity of human anti-NOR antibodies,a distinct species of "natural" anti-alpha-galactosyl antibodies

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Galalpha1,3Gal antibodies, alpha-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and over 100 times less strongly with Galalpha1-4GalNAc (NOR-di). The antibodies reacted also with Galalpha1-4Gal and Galalpha1-4Galbeta1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Galalpha1,3Gal antibodies reacted most strongly with Galalpha1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested alpha-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-alpha-galactosyl antibodies with high affinity for the Galalpha1-4GalNAcbeta1-3Gal sequence present in rare NOR erythrocytes.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

Production and characterization of transgenic mice systemically expressing endo-beta-galactosidase C

The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alphaGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta1,4-galactosyltransferase 1 (beta4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.     

Mice lacking alpha1,3-fucosyltransferase IX demonstrate disappearance of Lewis x structure in brain and increased anxiety-like behaviors

The 3-fucosyl-N-acetyllactosamine [Lewis x (Le(x)), CD15, SSEA-1] carbohydrate structure is expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth, and neuronal migration during development. To characterize the functional role of Le(x) carbohydrate structure in vivo, we have generated mutant mice that lack alpha1,3-fucosyltransferase IX (Fut9(-/-)). Fut9(-/-) mice were unable to synthesize the Le(x) structure carried on glycoproteins and glycolipids in embryonic and adult brain. However, no obvious pathological differences between wild-type and Fut9(-/-) mice were found in brain. In behavioral tests, Fut9(-/-) mice exhibited increased anxiety-like responses in dark-light preference and in elevated plus maze tests. Immunohistochemical analysis showed that the number of calbindin-positive neurons was decreased in the basolateral amygdala in Fut9(-/-) mice. These observations indicated that the carbohydrates synthesized by Fut9 play critical roles in functional regulations of interneurons in the amygdalar subdivisions and suggested a role for the Le(x) structure in some aspects of emotional behavior in mice.     

Recent intron gain in elongation factor-1alpha of colletid bees (Hymenoptera: Colletidae)

We discovered the presence of a unique spliceosomal intron in the F1 copy of elongation factor-1alpha (EF-1alpha) restricted to the bee family Colletidae (Hymenoptera: Apoidae). The intron ranges in size from 101 to 1044 bp and shows no positional sliding. Our data also demonstrate the complete absence of this intron from exemplars representing all other bee families, as well as from close hymenopteran relatives. A review of the literature finds that this intron is likewise absent from all other arthropods for which data are available. This provides unambiguous evidence for a relatively recent intron insertion event in the colletid common ancestor and, at least in this specific instance, lends support to the introns-late hypothesis. The comparative distribution of this novel intron also supports the monophyly of Colletidae and the exclusion of the Stenotritidae from this family, providing an example of the potential of some introns to act as robust markers of shared descent.     

Immobilized Marasmius oreades agglutinin: use for binding and isolation of glycoproteins containing the xenotransplantation or human type B epitopes

The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins.     

Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified alpha-mannosidase from Candida albicans

A soluble alpha-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS-polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2) isomer B and mannose as a function of time of incubation up to 12 h at 37 degrees C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man(7)GlcNAc(2) and probably Man(6)GlcNAc(2). These two products were also observed when Man(8)GlcNAc(2) isomer B instead of Man(9)GlcNAc(2) was used as substrate. Other oligosaccharides, such as Man(6)GlcNAc(2)-Asn, Man(5)GlcNAc(2)-Asn, and the alpha1,3- and alpha1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an alpha1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.     

Molecular cloning and functional expression of a novel Helicobacter pylori alpha-1,4 fucosyltransferase

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is associated with gastric cancer and lymphoma. This microorganism synthesizes fucosylated oligosaccharides, predominantly the Galb-1,4GlcNAc (Type II) blood group antigens Lewis X and Y, whereas a small population also expresses the Galb-1,3GlcNAc (Type I) blood group antigens Lewis A and B. These carbohydrate structures are known to mimic host cell antigens and permit the bacteria to escape from the host immune response. Here, we report the cloning and characterization of a novel H. pylori alpha-1,4 fucosyltransferase (FucT). In contrast to the family members characterized to date, this enzyme shows exclusively Type I acceptor substrate specificity. The enzyme consisting of 432 amino acids (MW 50,502 Da) was cloned using a polymerase chain reaction (PCR)-based approach. It exhibits a high degree of identity (75-87%) and similar structural features, for example, in the heptamer repeat pattern, with other H. pylori FucTs. The kinetic characterization revealed a very efficient transferase (k(cat)/Km = 229 mM(-1) s(-1)) for the Type I acceptor substrate (Gal)-1,3 GlcNAc-Lem (1). Additionally, the enzyme possesses a broad tolerance toward nonnatural Type I acceptor substrate analogs and therefore represents a valuable tool for the chemoenzymatic synthesis of Lewis A, sialyl Lewis A as well as mimetics thereof.     

Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.     

Carbohydrate residues downstream of the terminal Galalpha(1,3)Gal epitope modulate the specificity of xenoreactive antibodies

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galalpha(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional alpha1,3-galactosyltransferase (alpha1,3GT), which transfers alphaGal to N-acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Galalpha(1,3)Gal is still expressed in alpha1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in alpha1,3GT-/- mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid-linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface-expressed Galalpha(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three-dimensional models suggest that the terminal alpha-linked Gal binds tightly into the antibody-binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.     

Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.     

Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.     

Identification of duplicated fourth alpha2-adrenergic receptor subtype by cloning and mapping of five receptor genes in zebrafish

The alpha(2)-adrenergic receptors (alpha(2)-ARs) belong to the large family of rhodopsinlike G-protein-coupled receptors that share a common structure of seven transmembrane (TM) alpha-helices. The aims of this study were (1) to determine the number of alpha(2)-AR genes in a teleost fish, the zebrafish (Danio rerio), (2) to study the gene duplication events that generated the alpha(2)-AR subtypes, and (3) to study changes in receptor structure that have occurred since the divergence of the mammalian and fish lineages. Here, we report the cloning and chromosomal mapping of fish orthologs for all three mammalian alpha(2)-ARs. In addition, we identified a fourth alpha(2)-AR subtype with two duplicates in zebrafish. Chromosomal mapping showed that the zebrafish alpha(2)-AR genes are located within conserved chromosomal segments, consistent with the origin of the four alpha(2)-AR subtypes by two rounds of chromosome or block duplication before the divergence of the ray fin fish and tetrapod lineages. Thus, the fourth subtype has apparently been present in the common ancestor of vertebrates but has been deleted or is yet to be identified in mammals. The overall percentage identity between the fish and mammalian orthologs is 53% to 67%, and in the TM regions 80% to 87%. These values are clearly lower than what is observed between mammalian orthologs. Still, all of the residues thought to be important for alpha(2)-adrenergic ligand binding are conserved across species and subtypes, and even the most divergent regions of the fish receptors show clear "molecular fingerprints" typical for orthologs of a given subtype.     

A specific detection of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins based on the specificity of N-acetylglucosaminyltransferase VI

Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface-in particular, the production of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5'-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcbeta1-6Manalpha1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto alpha1-acid glycoprotein (AGP), which is known to contain GlcNAcbeta1-6Manalpha1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcbeta1-6Manalpha1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and beta-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS-PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L(4)-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L(4)-PHA.     

Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth,apoptosis, and gene expression in 3LL Lewis lung carcinoma cells

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and beta1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF alpha receptor mRNA and protein, but in the SM3-reconstituted cells PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.