Α-galactosidase Treatment Research Articles

Binding of Mouse Sperm to β-Galactose Residues on Egg Zona Pellucida and Asialofetuin-Coupled Beads

Mouse eggs fixed with paraformaldehyde were incubated with various exoglycosidases and their sperm-binding activities were examined. The number of sperm bound per egg was increased by sialidase treatment and decreased by β-galactosidase treatment. No prominent reduction of sperm-binding was observed after α-galactosidase treatment. Mouse sperm also bound to asialofetuin-coupled agarose beads but not to fetuin-coupled beads. The sperm-binding was abolished when asialofetuin-gel was treated with β-galactosidase specific to the β1→4 linkage orN-Glycanase. Furthermore asialofetuin, but not β-galactosidase-treated asialofetuin, competitively inhibited the binding of sperm to the zona pellucida of live eggs. These results suggest that mouse sperm recognize β-galactose residues of the zona pellucida at the initial stage of the binding.

Enzymatic degradation of oligosaccharides in soybean flours

The oligosaccharide content was determined in the flour of six soybean cultivars grown in India. The effect of cooking and soaking on the removal of oligosaccharides from the whole dry seeds was also studied. Crude α-galactosidase treatment on soybean flour reduced the raffinose and stachyose contents by 90.4% and 91.9%, respectively. This information could stimulate interest in the large-scale production of oligosaccharide-free soybean flour, by modulating its flatulence-causing properties.

Xenotransplant issues

To determine the role of the terminal α-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean α-galactosidase. A practically complete removal of terminal α-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the α-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the α-galactosyl residues. When α-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD 16 NK cells is dependent on IgG1 and IgG3, and not IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and α-galactosidasetreated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon α-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize α-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the α-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts. Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen. were in the minority and mainly located in the peripheral parts of the ICC xenograf. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 doublepositive cells were found to be large and more granular than the CD4 single-positive cells No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (=15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive β2- microglobulin-deficient mutant, C4.4–25, lumphoma cell line EL-4, served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but readily rejected the NK cell-sensitive lymphoma cell line. taken together, ICC xenograft rejection in mice seems to be T Cell-dependent, as evidenced in the nude mice model, while the main effector cel appears to be a macrophage with a unique phenotype.

Quantitation of the rabbit intestinal glycolipid receptor for Shiga toxin: Further evidence for the developmental regulation of globotriaosylceramide in microvillus membranes

Shiga toxin, produced by Shigella dysenteriae 1, causes enterotoxic, cytotoxic, and neurotoxic effects, which may be mediated by a glycolipid receptor, globotriaosylceramide, Gb3. To study the relationship of this receptor and toxin effects, globotriaosylceramide was quantitated and further characterized in rabbit small intestinal microvillus membranes at various ages. Glycolipids were extracted from rabbit microvillus membranes, purified on Unisil columns, and quantitated by highperformance liquid chromatography. The major glycolipid peaks were hydroxylated fatty acid-containing glucosylceramide, lactosylceramide, and globotriaosylceramide. There was a marked increase of globotriaosylceramide levels with age, ranging from 0.02 to 16.2 pmol/μg microvillus membrane protein in neonates and adults, respectively. The globotriaosylceramide peak was susceptible to α-galactosidase treatment, which produced an elevation in the lactosylceramide peak, but markedly reduced globotriaosylceramide content in 34-day-old rabbits. Binding of iodinated Shiga toxin to globotriaosylceramide was documented on highperformance thin-layer chromatography plates by autoradiography. The glycolipid receptor for Shiga toxin in rabbit microvillus membranes is thus a hydroxylated fatty acid-containing globotriaosylceramide. This moiety is virtually absent in neonates and gradually increases with age. Quantitative differences in globotriaosylceramide may be the underlying basis for the age-specific differences in functional responsiveness of rabbit intestinal tissue to Shiga toxin.

Isolation and Characterization of the α-Galactosidase of Waste Lager Yeast (Saccharomyces carlsbergensis)

α-Galactosidase treatment has been suggested as a means of removing unwanted α-galactoside sugars from legume products and from sugar beet molasses. Waste lager yeast (Saccharomyces carlsbergensis, also known as S. uvarum) was found to contain 27 units of α-galactosidase/g yeast cells (dry weight). α-Galactosidase activity was separated into 3 active peaks by DEAE cellulose chromatography. Subsequent gel filtration indicated molecular weights of the α-galactosidases in peaks A, B and C to be 118,000 daltons, 95,000 daltons and 65,000 daltons respectively. The isoelectric point of all 3 forms of the enzyme was 4.4±0.1. The enzyme of peak C had a pH optimum of 4.5. Km values ( ± standard errors) were 2.58±0.11mM p-nitrophenyl α-D-galactopyranoside (PNPG) and 16.7±1.2mM raffinose.

Mass spectrometric analysis of permethylated glycosphingolipids II. Comparative studies on different blood-group active and related erythrocyte membrane glycosphingolipids

Three isomeric ceramide tetrasaccharides — P blood-group active globoside, lacto-N-neotetraosyl ceramide as ABH blood-group precursor, both isolated from human erythrocytes and “asiologanglioside” from human brain as reference standard — and two ceramide pentasaccharides — H blood-group active glycosphingolipid, obtained from blood-group B active ceramide hexasaccharide of human B erythrocytes after α-galactosidase treatment and ceramide pentasaccharide from rabbit erythrocytes with B-like blood-group activity — were investigated by mass spectrometry after permethylation. The carbohydrate moiety exhibits differences not only concerning the sugar sequence but also with regard to the position of some glycosidie linkages: Oligosaccharides containing N-acetylhexosamine substituted at position 4 produce spectra that are distinctly different from those containing C-3 substituted N-acetylhexosamines, thus allowing the differentiation between type 1 and type 2 carbohydrate chains. Moreover, oligosaccharide ions with a hexose at the cleavage site exhibit a fragmentation pattern different from those with a N-acetylhexosamine at the “reducing terminal”. The intensity ratio between parent ion and parent ion — 32 mass units is Q ⩾ 3 in the first case, whereas in the latter case Q is <1. The Q-values are given for 14 oligosaccharide ions. Differences in the composition of the ceramide residues can also be deduced from the mass spectra.

Action de l'α-galactosidase du café sur quelques galactomannanes

Coffee-bean α-galactosidase (3.2.1.22) splits, at similar initial rates, α- D-galactosyl units from various galactomannans isolated from Trifolium repens, Genista scoparia, Gleditschia ferox, Gleditschia triacanthos, and Ceratonia siliqua. Except for white-clover, genista, and carob-tree galactomannans, α-galactosidase treatment resulted in insoluble mannan-like polysaccharides. A water-insoluble fraction containing 24 residues of mannose for each residue of galactose was obtained from the galactomannan of Gleditschia ferox. It contains chains having a β- D-(1 → 4)-mannopyranosyl repeating unit. These chains have probably a few side-branches. The few remaining D-galactose residues are linked (1 → 6) as side-branches to the D-mannose chains.