Accelerated proliferation and abnormal differentiation of epidermal keratinocytes in endo-beta-galactosidase C transgenic mice

Transgenic (TG) mice that have systemically expressed endo-beta-galactosidase C (EndoGalC) have rough and flaky skin. This skin phenotype is detectable around 5 days postnatal and becomes obscure by 2 weeks after birth. Their epidermis is thickened but the dermis and hair follicles are normal in structure. EndoGalC, which removes the terminal Galalpha1-3Gal disaccharide (alphaGal epitope), was expressed in the epidermis of TG mice. GS-IB4 lectin staining showed that the alphaGal epitope did not exist in the epidermis in TG but existed in wild-type (WT) mice. In TG mice, N-acetylglucosamines were exposed by EndoGalC, which is detected using GS-II lectin. To understand the cause of the epidermal thickening and skin phenotype, we examined the proliferation and differentiation of kerationocytes. BrdU-pulse-labeling revealed that proliferating keratinocytes increased approximately three-fold in TG epidermis compared to WT one. In TG epidermis, the expression domain of cytokeratin 14 increased from 1-2 layers to 4-5 layers and co-expressed with cytokeratin 6 and 10 in the upper layers. The layers expressing involucrin and loricrin also increased but those expressing filaggrin and transglutaminase looked normal. The localization of E-cadherin was similar in both TG and WT mice. Although TG mice showed delayed development of the barrier function around 8 days postnatal, they acquired the function by 12 days after birth. These results suggest that the absence of the alphaGal epitope or the exposed N-acetylglucosamine terminal could play a critical role in the proliferation of basal keratinocytes and differentiation of them into the spinous cells in newborn mice.     

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.     

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.     

Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney

Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.     

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.     

Naturally occurring anti-alpha-galactosyl antibodies: relationship to xenoreactive anti-alpha-galactosyl antibodies.

Antibodies produced by an individual without a known history of sensitization to the relevant antigen are called "natural" antibodies. Some natural antibodies, called xenoreactive antibodies, react with the cells of foreign species. Most xenoreactive antibodies in humans and higher primates bind to a nonreducing terminal galactose expressed by pigs and other lower mammals. Although human natural antibodies which bind to one or more of a variety of terminal alpha-galactosyl structures have been identified previously, the antigen recognized by anti-alpha-galactosyl antibodies on the cells of foreign species is thought to be exclusively Galalpha1-3Gal. Thus, anti-alpha-galactosyl antibodies which do not react with Galalpha1-3Gal are thought to be nonxenoreactive. Here, we identify natural antibodies in human serum which bind to Galalpha1-6Hexosepyrranosides but not Galalpha1-3Gal, indicating that these antibodies are not xenoreactive. Various lower mammals were found to have natural anti-Galalpha1-2Gal antibodies in their sera, suggesting that at least some anti-Galalpha1-2Gal antibodies might not be xenoreactive and indicating, surprisingly, that anti-alpha-galactosyl antibodies are much more phylogenetically disperse than previously known. Also surprising was the finding that some natural antibodies which bind to Galalpha1-3Gal in vitro do not bind to porcine xenografts. These studies show that naturally occurring anti-alpha-galactosyl antibodies in mammalian serum include antibodies with a greater variety of reactivities than previously thought, only some of which would bind to a porcine xenograft. Further, these studies show that the methods used to detect anti-alpha-galactosyl antibodies of relevance in xenotransplantation must be carefully evaluated to avoid detection of anti-alpha-galactosyl antibodies which would not bind to a porcine organ and which therefore are not involved in xenograft rejection.     

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.     

Mapping the expressed glycome and glycosyltransferases of zebrafish liver cells as a relevant model system for glycosylation studies

The emergence of zebrafish as a model organism for human diseases was accompanied by the development of cellular model systems that extended the possibilities for in vitro manipulation and in vivo studies after cell implantation. The exploitation of zebrafish cell systems is, however, still hampered by the lack of genomic and biochemical data. Here, we lay a path toward the efficient use of ZFL, a zebrafish liver-derived cell system, as a platform for studying glycosylation. To achieve this, we established the glycomic profile of ZFL by a combination of mass spectrometry and NMR. We demonstrated that glycoproteins were substituted by highly sialylated multiantennary N-glycans, some of them comprising the unusual zebrafish epitope Galβ1-4[Neu5Ac(α2,3)]Galβ1-4[Fuc(α1,3)]GlcNAc, and core 1 multisialylated O-glycans. Similarly, these analyses established that glycolipids were dominated by sialylated gangliosides. In parallel, analyzing the expression patterns of all putative sialyl- and fucosyltransferases, we directly correlated the identified structures to the set of enzymes involved in ZFL glycome. Finally, we demonstrated that this cell system was amenable to metabolic labeling using functionalized monosaccharides that permit in vivo imaging of glycosylation processes. Altogether, glycomics, genomics, and functional studies established ZFL as a relevant cellular model for the study of glycosylation.     

Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Analysis of human serum antibody-carbohydrate interaction using biosensor based on surface plasmon resonance

Surface plasmon resonance (SPR) was used to monitor the interaction of alphaGal-antibodies from human blood group O serum with linear blood group B-saccharides, employing Galalpha1-3Galbeta1-4GlcNAc-HSA immobilised on a sensor chip surface. Strong binding of antibodies, as evident from high relative response values exceeding 200 RU, was observed. The interaction was influenced by the nature of the oligosaccharide that was added to the antibody sample prior to measurement. For example, the addition of either of the linear B-saccharides Galalpha1-3Gal and Galalpha1-3Galbeta1-4GlcNAc produced complete inhibition of antibody binding to the sensor surface, whereas the addition of the related but non-specific blood group A saccharide, GalNAcalpha1-3(Fucalpha1-2)Gal, had little effect on binding. The technique was used for the rapid monitoring of the removal of alphaGal-antibodies from human serum by affinity columns, which contained either Galalpha1-3Gal or Galalpha1-3Galbeta1-4GlcNAc as ligand. The above carbohydrates are currently evaluated as inhibitors or as affinity ligands, in the prevention of hyperacute rejection during xenotransplantation.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.     

Characterization of the rat alpha(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Galalpha(1,3)Gal by two separate glycosylation pathways

The important xenoepitope Galalpha(1,3)Gal was thought to be exclusively synthesized by a single alpha(1,3)galactosyltransferase. However, the cloning of the distant family member rat iGb3 synthase, which is also capable of synthesizing Galalpha(1,3)Gal as the glycolipid structure iGb3, challenges the notion that alpha(1,3)galactosyltransferase is the sole Galalpha(1,3)Gal-synthesizing enzyme. We describe the cloning of the rat homolog of alpha(1,3)galactosyltransferase, showing that indeed the rat expresses two distinct alpha(1,3)galactosyltransferases, alpha(1,3)GT and iGb3 synthase. Rat alpha(1,3)galactosyltransferase shows a high amino acid sequence identity with the alpha(1,3)galactosyltransferase of mouse (90%), pig (76%), and ox (75%), in contrast to the low amino acid sequence identity (42%) with iGb3 synthase. The rat alpha(1,3)galactosyltransferase is expressed in heart, brain, spleen, kidney, and liver and has a similar intron/exon structure to the mouse alpha(1,3)galactosyltransferase. Transfection studies show that in contrast to the iGb3 synthase, rat alpha(1,3)galactosyltransferase can synthesize Galalpha(1,3)Gal on glycoproteins but cannot synthesize the glycolipid iGb3, defining two separate glycosylation pathways for the synthesis of Galalpha(1,3)Gal. Furthermore iGb3 synthase was found to be distinct from alpha(1,3)GT with its ability to synthesize poly-alpha-Gal glycolipid structures.     

Xenotransplantation: in vitro analysis of synthetic alpha-galactosyl inhibitors of human anti-Galalpha1-->3Gal IgM and IgG antibodies

Pig-to-human xenotransplantation might be an option to overcome the increasing shortage of human donor organs. However, naturally occurring antibodies in human blood against the Galalpha1-->3Gal antigen on pig endothelial cells lead to hyperacute or, if prevented, acute or delayed vascular rejection of the pig graft. The purpose of this study was therefore to evaluate synthetic oligosaccharides with terminal Galalpha1-->3Gal to inhibit antigen-binding and cytotoxicity of anti-alphaGal antibodies against pig cells. Different oligosaccharides were synthesized chemically and by a combined chemico-enzymatic approach. These included monomeric di-, tri-, and pentasaccharides, a polyacrylamide-conjugate (PAA-Bdi), as well as di-, tetra-, and octamers of Galalpha1-->3Gal. All were tested for inhibitory activity by anti-alphaGal ELISA and complement-dependent cytotoxicity tests. PAA-Bdi was the best inhibitor of binding as well as cytotoxicity of anti-alphaGal antibodies. Monomeric oligosaccharides efficiently prevented binding of anti-alphaGal IgG, but less well that of anti-alphaGal IgM, with tri- and pentasaccharides showing a better efficacy than the disaccharide. The two trisaccharides Galalpha1-->3Galbeta1-->4GlcNAc and Galalpha1-->3Galbeta1-->3GlcNAc were equally effective. Oligomers of Galalpha1-->3Gal were more effective than monomers in blocking the binding of anti-alphaGal IgG. However, they could not block IgM binding, nor could they match the efficacy of PAA-Bdi. We conclude that oligosaccharides with terminal Galalpha1-->3Gal, most effectively as PAA-conjugates, can prevent binding and cytotoxicity of human anti-alphaGal in vitro. The PAA-Bdi conjugate might be most suited for use as a Sepharose-bound immunoabsorption material.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen

In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.     

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.     

Isolation and characterization of major glycoproteins of pigeon egg white: ubiquitous presence of unique N-glycans containing Galalpha1-4Gal

Ovotransferrin (POT), two ovalbumins (POA(hi) and POA(lo)), and ovomucoid (POM) were isolated from pigeon egg white (PEW). Unlike their chicken egg white counterparts, PEW glycoproteins contain terminal Galalpha1-4Gal, as evidenced by GS-I lectin (specific for terminal alpha-Gal), anti-P(1) (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) monoclonal antibody, and P fimbriae on uropathogenic Escherichia coli (specific for Galalpha1-4Gal). Galalpha1-4Gal on PEW glycoproteins were found in N-glycans releasable by treatment with glycoamidase F. The respective contents of N-glycans in each glycoprotein were 3.5%, POT; 17%, POA(hi); and 31-37%, POM. POA(hi) has four N-glycosylation sites, in contrast to chicken ovalbumin, which has only one. High performance liquid chromatography analysis showed that N-glycans on POA(hi) were highly heterogeneous. Mass spectrometric analysis revealed that the major N-glycans were monosialylated tri-, tetra-, and penta-antennary oligosaccharides containing terminal Galalpha1-4Gal with or without bisecting N-acetylglucosamine. Oligosaccharide chains terminating in Galalpha1-4Gal are rare among N-glycans from the mammals and avians that have been studied, and our finding is the first predominant presence of (Galalpha1-4Gal)-terminated N-glycans.     

Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B). The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B). The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal. Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.     

Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity

Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Galalpha(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in alpha(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.     

Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.