Characterization of a UDP-Gal:Galβ1–3GalNAc α1,4-Galactosyltransferase Activity in a Mamestra brassicaeCell Line

N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139 -146). Twentyfive major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Gal␣(1,4)Gal␤(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. NAcetylneuraminic acids were ␣(2,6)-linked only to ␤-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.The presence of galabiose (Gal␣4Gal) sequence in N-glycans in any animal is only rarely noted. The only documented presence of such a sequence in N-glycan was that found in turtle dove ovomucoid (1, 2). The only other recorded presence of galabiose sequence were both in the O-glycosides found in the Chinese swiftlet nest (3) and in an insect cell line Mamestra brassicae clone SPCMb-92-C6 (4). As shown in the preceding paper (5), we have demonstrated that galabiose sequence is abundantly present in pigeon egg white (PEW) 1 glycoproteins, and it always occupies the terminal positions of the multiantennary structures. In the major N-glycans from the pigeon ovalbumin, no sialylation is found on the branch that contains the galabiose sequence. In contrast, chicken ovalbumin contained mostly high mannose type and hybrid types (6, 7) whereas chicken ovomucoid contains mostly galactose-less complex type (8). The preceding paper (5) also notes that, in contrast to the chicken and other avian egg whites hitherto investigated, the N-glycans in pigeon egg white glycoproteins seem to be more "mature," containing more of the multiantennary complex type structures. In view of these unique structural features, we felt that detailed structural studies were in order, not only to understand the underlying evolutionary sig...

supporting

confidence: 67%

N-Glycan Structures from the Major Glycoproteins of Pigeon Egg White

Takahashi

Khoo

Suzuki

et al. 2001

“…teins expressed in insect cells, although variable, show mostly high mannose-type (Man 5-9 -GlcNAc 2 ) and short truncated structures with fucose ␣1,6-linked to the asparagine-bound GlcNAc residue (35). The binding of ConA to mannose does not distinguish among different manno-oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Wang

Sun

Al-Qamari

et al. 2001

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant ␣ 5 and ␤ 1 proteins and ␣ 5 ␤ 1 integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of ␣ 5 ␤ 1 to fibronectin. Co-immunoprecipitation of GT1b and ␣ 5 ␤ 1 from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified ␣ 5 ␤ 1 from SCC12 cells and insect recombinant ␣ 5 ␤ 1 , particularly the ␣ 5 subunit, further suggest interaction between ganglioside and ␣ 5 ␤ 1 . The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of ␣ 5 ␤ 1 from SCC12 and insect cells or poorly glycosylated recombinant ␣ 5 ␤ 1 from Escherichia coli cells. The GT1b-␣ 5 ␤ 1 interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in ␣ 5 ␤ 1 . The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate ␣ 5 ␤ 1 -mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the ␣ 5 subunit of ␣ 5 ␤ 1 integrin. Human keratinocyte motility on a fibronectin (FN)1 matrix is critical for the re-epithelialization of healing wounds, for the spread of cutaneous malignancy, and in cutaneous embryogenesis. Although the molecular events that influence this migration are poorly understood, the interaction between keratinocyte ␣ 5 ␤ 1 integrin and the arginine-glycine-aspartic acid (RGD) site of FN is known to be key (1). Increased expression of ␣ 5 ␤ 1 has been shown in keratinocytes at the migrating edge of wounds in vivo, in lesional skin of patients with psoriasis, and in cultured keratinocytes (2-4). Both receptor clustering on keratinocytes and ligand occupancy of ␣ 5 ␤ 1 are required to activate intracellular signal transduction components (5), including focal adhesion kinase, phosphatidylinositol 3-kinase, protein kinase C, and integrin-linked kinase, leading to cell adhesion to .Gangliosides are glycosphingolipids characterized by the presence of one or more sialic acid moieties in the oligosaccharide chain (9). The role of gangliosides, which are localized to the outer leaflet of the plasma membrane of eukaryotic cells, is largely unknown, but studies with cultured keratinocytes and keratinocyte-derived cells suggest that gangliosides are involved in regulating cellular proliferation, differentiation, and adhesion (10 -13). The discovery that gangliosides inhibit cell attachment and spreading on a FN matrix (14) led investigators to consider gangliosides to be the cell receptors for FN before integrin ␣ 5 ␤ 1 was identified (15,16). Although these early studies showed that the terminal sialic acid residues of gangliosides were critica...

“…Both enzymes transfer to galactose, but, whereas the acceptor disaccharide specificity of the ␣4Gl-cNAc-T appears to be broad, ␣4Gal-T1 is apparently highly specific for the glycolipid, lactosylceramide. Lopez et al (44) recently characterized an ␣4Gal-T activity in insect cells and found it had preferred acceptor substrate specificity for Gal␤1-3GalNAc␣1-R rather than lacto-series structures. Thus, the acceptor substrate specificity is similar to that of the ␣4Glc-NAc-T and different from ␣4Gal-T1.…”

Section: Fig 4 Expression Of Full Coding ␣4gal-t1 Variants In Highmentioning

confidence: 99%

Cloning and Expression of the Histo-blood Group PkUDP-galactose:Galβ1–4Glcβ1-Cer α1,4-Galactosyltransferase

Steffensen¹,

Carlier²,

Wiels³

et al. 2000

117

137

The molecular genetic basis of the P histo-blood group system has eluded characterization despite extensive studies of the biosynthesis of the P 1 , P, and P k glycolipids. The main controversy has been whether a single or two distinct UDP-Gal:Gal␤1-R 4-␣-galactosyltransferases catalyze the syntheses of the structurally related P 1 and P k antigens. The P 1 polymorphism is linked to 22q11.3-ter. Data base searches with the coding region of an ␣4GlcNAc-transferase identified a novel homologous gene at 22q13.2 designated ␣4Gal-T1. Expression of full coding constructs of ␣4Gal-T1 in insect cells revealed it encoded P k but not P 1 synthase activity. Northern analysis showed expression of the transcript correlating with P k synthase activity and antigen expression in human B cell lines. Transfection of P k -negative Namalwa cells with ␣4Gal-T1 resulted in strong P k expression. A single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p phenotype, confirming that ␣4Gal-T1 represented the P k gene. Sequence analysis of the coding region of ␣4Gal-T1 in P 1 ؉/؊ individuals did not reveal polymorphisms correlating with P 1 P 2 typing.The P histo-blood group system is the last of the known carbohydrate defined blood group systems for which the molecular genetic basis has not yet been clarified. The P blood group system involves two major blood group phenotypes, P 1 ϩ and P 1 Ϫ, with approximate frequencies of 80% and 20%, respectively (1, 2). P 1 Ϫ individuals normally express the P antigen (P 1 Ϫ is designated P 2 when P antigen expression is demonstrated), but the rare P k phenotype lacks the P antigen, while the rare p phenotype lack both P and P k antigens (for reviews, see Refs. 3-7). The P 1 ϩ phenotype is defined by expression of the neolacto-series glycosphingolipid P 1 (for structures, see Table I) (8). In contrast, the P, P k , and p antigens constitute intermediate steps in biosynthesis of globo-series glycolipids and give rise to P 1 k , P 2 k , and p phenotypes (9). Although the rare P k phenotype shows the same frequency of P 1 antigen expression as individuals expressing the P antigen, the p phenotype is always associated with lack of P 1 antigen expression. Extensive studies of the chemistry, biosynthesis, and genetics of the P blood group system identified the antigens as being exclusively found on glycolipids, with the blood group specificity being synthesized by at least two distinct glycosyltransferase activities; UDP-galactose:␤-D-galactosyl-␤1-R 4-␣-Dgalactosyltransferase (␣4Gal-T) 1 activity(ies) for P k and P 1 syntheses and UDP-GalNAc:Gb 3 3-␤-N-acetylgalactosaminyltransferase activity (EC 2.4.1.79) for P synthesis (for reviews, see Refs. 6 and 7). At least two independent gene loci, P and P 1 P k , are involved in defining these antigens. The P blood group-associated LKE antigen, shown to be the extended sialylated Gal-globoside structure (10), may involve polymorphism in an ␣2,3-sialyltransferase activity.A long-standing controversy has been whether a single...