Terminal Galactosyl Residues Research Articles

Purification and characterization of a thermostable Galium aparine β-galactosidase: A competent agent with enhanced cytotoxic activity against MCF-7 cell line

β-galactosidases (EC 3.2.1.23) are capable of catalysing two distinct types of reactions: transgalactosylation and hydrolysis. It is very important for the senescence of cancer cells. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from various sources including plants, animals, bacteria, yeasts, and filamentous fungi. Purification and thermodynamic characterization of β-galactosidase from Galium aparine seeds, and in vitro efficacy assessment of a breast cancer cell line. The enzyme was purified by using ammonium sulphate precipitation, dialysis, DEAE cellulose, and Sephadex G-100 chromatography. The enzyme was purified to 6.4 fold with a yield of 14 % and specific activity of 11.6 U/mg of protein. SDS-PAGE and MALDI-TOF analysis revealed that β-galactosidase is a monomer with a molecular weight of 35 kDa. The optimum pH and temperature for the purified enzyme were 6.0 and 50 ºC. The enzyme had Km and Vmax values of 0.25 mM and 32 µmol/min, respectively. The thermodynamic parameters (ΔHº, ΔGº, ΔSº) for the irreversible denaturation of the enzyme were also investigated at different temperatures (50–75 ºC). Lactose in milk was successfully hydrolyzed using a purified enzyme at a rate of 0.0121 min−1 and 12.12 min. This is better than other studied β-galactosidases. These results suggest the use of β-galactosidase from G. aparine for producing delactosed milk for the lactose-intolerant population and prebiotic synthesis. pH and optimum temperature and its activation by Ca2+ shows that it is suitable for milk processing. As a result, the enzyme was stable even at higher temperatures and had the potential to undergo catalysis, making it economically viable, primarily in the food and pharmaceutical industries. The present study is aimed to purification and thermodynamic characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Розповсюдженість, властивості та практичне значення α-галактозидази

The achievements of modern enzymology have greatly expanded the possibilities of practical use of enzymes, primarily in medicine and the food industry, which is due to their advantages over chemical catalysts: selectivity and stereospecificity of action, the possibility of achieving high rates of conversion of substrates under relatively mild technological conditions, harmlessness to the environment and humans. The microbial producers have proven themselves as a convenient and economical source of biotechnologically important enzyme preparations of hydrolytic action. One of such enzymes is α-galactosidase, which has the ability to cleave terminal α-linked galactosyl residues from a wide range of natural and synthetic compounds. The review presents data on the spread of the enzyme among different groups of microorganisms and provides a comparative description of their catalytic properties. A range of issues related to the physiological role of the enzyme in plant and animal organisms, localization and functional properties in bacteria and fungi are outlined. The place of α-galactosidase of microorganisms in the modern hierarchical classification of glycosidases is shown. The areas of possible use of the enzyme as an efficient tool for the modification of oligo- and polysaccharides in various industries are described.

Structure Flexibility of Alpha-galactosidase from a Marine Psychrophilic Yeast, Glaciozyma antarctica PI12

Factors that contribute to maintaining the flexibility or stability of an enzyme structure may depend on the composition of each amino acid with different characteristics, providing a purpose and bonding features within the structure. Based on this assumption, a study using homology modeling and a comparative study to observe different structure behaviors of an enzyme at an extremely low temperature (psychrophile) against temperate (mesophile) and high temperature (thermophile) was performed. The subject, a-galactosidase from Glaciozyma antarctica as a marine psychrophilic candidate was chosen against a-galactosidase from Trichoderma reesei (mesophile) and Ramsonia emersonii (thermophile). This enzyme catalyzes the hydrolysis of a-1-6 linked terminal galactosyl residues which can be found in a wide range of the organism. The ability of G. antarctica to grow in extremely cold temperatures rendered the question that the enzyme must have special characteristics to adapt to the cold condition. Based on the homology modeling and molecular dynamics study, a comparison of the structure of G. antarctica a-galactosidase enzymes with its homolog from the mesophilic and thermophilic fungi showed that G. antarctica a-galactosidase enzyme confers its flexibility by the increased number of small amino acids with reduced charges, more loops, a fewer number of hydrogen and disulfide bonds in its structure. Furthermore, a-galactosidase has potential for commercialization in bleach paper and the baking industry also a treatment for bloating and Fabry disease.

Purification and characterization of β-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7).

β-galactosidases (EC 3.2.1.23) are able to catalyze two different types of reactions, namely hydrolysis and transgalactosylation. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from microbial sources including bacteria, yeasts, and filamentous fungi. The enzyme was purified from the crude enzyme using ammonium sulfate precipitation, dialysis, ion exchange chromatography using DEAE cellulose, fast protein liquid chromatography and high performance liquid chromatography. The enzyme was purified with 10.78 -fold with specific activity of 62U/mg of protein and yield of 28.26%. Molecular weight of β -galactosidase as estimated by using SDS-PAGE was 42kDa. Kinetic parameters Km and Vmax for purified enzyme were 0.48 and 0.96 respectively. Further the characterization and kinetic studies of purified enzyme were carried out. The optimum pH and temperature for maximum β-galactosidase activity were found to be 6, 40°C, respectively. The present study is aimed to purification, characterization and in vitro efficacy assessment in breast cancer cell line. The β-galactosidase isolated from Aspergillus terreus was found to be effective in the proliferation of MCF-7 breast cancer cells in vitro. The present study is aimed to purification and characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Synthesis and Characterization of Sialylated Lactose- and Lactulose-Derived Oligosaccharides by Trypanosoma cruzi Trans-sialidase.

Sialylated oligosaccharides contribute 12.6–21.9% of total free oligosaccharides in human milk (hMOS). These acidic hMOS possess prebiotic properties and display antiadhesive effects against pathogenic bacteria. Only limited amounts of sialylated hMOS are currently available. The aim of our work is to enzymatically synthesize sialylated oligosaccharides mimicking hMOS functionality. In this study, we tested mixtures of glucosylated-lactose (GL34), galactosylated-lactulose (LGOS), and galacto-oligosaccharide (Vivinal GOS) molecules, as trans-sialylation acceptor substrates. The recombinant trans-sialidase enzyme from Trypanosoma cruzi (TcTS) was used for enzymatic decoration, transferring (α2→3)-linked sialic acid from donor substrates to nonreducing terminal β-galactopyranosyl units of these acceptor substrates. The GL34 F2 2-Glc-Lac compound with an accessible terminal galactosyl residue was sialylated efficiently (conversion degree of 47.6%). TcTS sialylated at least 5 LGOS structures and 11 Vivinal GOS DP3–4 compounds. The newly synthesized sialylated oligosaccharides are interesting as potential hMOS mimics for applications in biomedical and functional-food products.

Sialylation Is Dispensable for Early Murine Embryonic Development in Vitro.

The negatively charged nonulose sialic acid (Sia) is essential for murine development in vivo. In order to elucidate the impact of sialylation on differentiation processes in the absence of maternal influences, we generated mouse embryonic stem cell (mESC) lines that lack CMP‐Sia synthetase (CMAS) and thereby the ability to activate Sia to CMP‐Sia. Loss of CMAS activity resulted in an asialo cell surface accompanied by an increase in glycoconjugates with terminal galactosyl and oligo‐LacNAc residues, as well as intracellular accumulation of free Sia. Remarkably, these changes did not impact intracellular metabolites or the morphology and transcriptome of pluripotent mESC lines. Moreover, the capacity of Cmas −/− mESCs for undirected differentiation into embryoid bodies, germ layer formation and even the generation of beating cardiomyocytes provides first and conclusive evidence that pluripotency and differentiation of mESC in vitro can proceed in the absence of (poly)sialoglycans.

Structure of Arabidopsis thaliana FUT1 Reveals a Variant of the GT-B Class Fold and Provides Insight into Xyloglucan Fucosylation.

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a SN2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi.

The galactoside 2-α-L-fucosyltransferase FUT1 from Arabidopsis thaliana: crystallization and experimental MAD phasing.

The plant cell wall is a complex network of polysaccharides made up of cellulose, hemicelluloses and pectins. Xyloglucan (XyG), which is the main hemicellulosic component of dicotyledonous plants, has attracted much attention for its role in plant development and for its many industrial applications. The XyG-specific fucosyltransferase (FUT1) adds a fucose residue from GDP-fucose to the 2-O position of the terminal galactosyl residues on XyG side chains. Recombinant FUT1 from Arabidopsis thaliana was crystallized in two different crystal forms, with the best diffracting crystals (up to 1.95 Å resolution) belonging to the monoclinic space group P21, with unit-cell parameters a = 87.6, b = 84.5, c=150.3 Å, β = 96.3°. Ab initio phases were determined using a two-wavelength anomalous dispersion experiment on a tantalum bromide-derivatized crystal with data collected at the rising and descending inflection points of the Ta white line. An interpretable electron-density map was obtained after elaborate density modification. Model completion and structural analysis are currently under way.

Relationship between structure and immunological activity of an arabinogalactan from Lycium ruthenicum

An immunologically active arabinogalactan (LRGP3) was selectively degraded by acetolysis, mild acid hydrolysis and enzymatic digestion. After exo-α-l-arabinofuranosidase digestion, 56% of the arabinosyl chains were released. The resistant product (LRGP3-AF) had markedly increased complement fixating activities. The acid hydrolysis product (LRGP3-T) contained (1→3)-linked (17.6%), (1→6)-linked (23.1%), (1→3,6)-linked (30.1%) and terminal (29.2%) galactosyl residues, and its complement fixating activity was lower than that of LRGP3-AF. The side chains (Oligo-S) consisted of arabinose, galactose, and rhamnose in the molar ratios 16.8:1.4:1.0. The complement fixating activity of Oligo-S was weak, but Oligo-S had potent macrophage stimulation activity. Degradation of arabinosyl residues in LRGP3 decreased the macrophage stimulation activity, but the galactan backbone still expressed partial activity. The results demonstrated that the galactan backbone of the polymer might be essential for the expression of complement fixating activity and the arabinosyl side chains could be more responsible for the macrophage activation activity. There may be several structurally different active sites involved in the immunological activity of LRGP3.

The activity of serum beta-galactosidase in colon cancer patients with a history of alcohol and nicotine dependence: preliminary data

Beta-galactosidase (GAL) is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates through the sequential release of beta-linked terminal galactosyl residues. The stimulation of activity of exoglycosidases and other degradative enzymes has been noted in cancers as well as in alcohol and nicotine addiction separately. This is the first study to evaluate the activity of the serum senescence marker GAL in colon cancer patients with a history of alcohol and nicotine dependence, as a potential factor of worse cancer prognosis. The material was serum of 18 colon cancer patients and 10 healthy volunteers. Ten colon cancer patients met alcohol and nicotine dependence criteria. The activity of beta-galactosidase (pkat/ml) was determined by the colorimetric method. Comparisons between groups were made using the Kruskal-Wallis analysis and differences evaluated using the Mann-Whitney U test. Spearman's rank correlation coefficient was used to measure the statistical dependence between two variables. The activity of serum GAL was significantly higher in colon cancer patients with a history of alcohol and nicotine dependence, in comparison to colon cancer patients without a history of drinking/smoking (p=0.015; 46% increase), and the controls (p=0.0002; 81% increase). The activity of serum GAL in colon cancer patients without a history of alcohol/nicotine dependence was higher than the activity in the controls (p = 0.043; 24% increase). Higher activity of beta-galactosidase may potentially reflect the accelerated growth of the cancer, invasion, metastases, and maturation, when alcohol and nicotine dependence coincide with colon cancer. For a better prognosis of colon cancer, alcohol and nicotine withdrawal seems to be required.

Terminal N-Linked Galactose Is the Primary Receptor for Adeno-associated Virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.

Substrate Specificities of α-Galactosidase from Rice

The α-galactosidase from rice cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose and stachyose, but also liberated the terminal galactosyl residues attached O-6 position of the reducing-end mannosyl residue in mannobiose and mannotriose. In addition, the enzyme tore off the stubbed galactosyl residues attached inner-mannosyl residues in mannopentaose. It also could catalyze efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.

Lectin histochemistry of the glycoconjugates in conjunctival goblet cells

The distribution of O- and N-linked glycoconjugates in human conjunctival goblet cells was examined histochemically using biotinylated and fluorescence-labeled lectins simultaneously. Both peanut agglutinin and Erythrina cristagalli agglutinin, specific for O- and N-linked sugar chains, respectively, bound to the same goblet cell, which demonstrated that a conjunctival goblet cell produces and contains both types of glycoconjugates. Maackia amurensis lectin II, specific for sialic acid alpha 2, 3 galactose, bound to the goblet cells, while Sambueus nigra agglutinin, specific for sialic acid alpha 2, 6 galactose, did not. This observation suggested that the terminal galactosyl residue of the glycoconjugates is alpha 2, 3-sialylated in the human conjunctival goblet cells.

The structure of the L9 immunotype lipooligosaccharide from Neisseria meningitidis NMA Z2491

The lipooligosaccharide (LOS) from the Neisseria meningitidis prototype serogroup A strain NMA Z2491, an L9 immunotype LOS, was isolated and structurally characterized using glycosyl composition and linkage determination, mass spectrometry, and both 1- and 2-D nuclear resonance spectroscopy. The results show that the L9 LOS has an identical structure to that of an L4 LOS structure with the exception that it does not contain a sialic acid residue linked to position 3 of the lactoneotetraose terminal galactosyl residue. Further, two oligosaccharides are present in the Z2491 LOS preparation, OS1 and OS2. They differ from one another only in that OS2 contains an added glycine moiety, presumably at O-7 on the inner core Hep II residue. The structures of these oligosaccharides are as follows: [Display omitted] where R = H or Gly.

Active-site Mapping of a Populus Xyloglucan endo-Transglycosylase with a Library of Xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.

Site-directed mutagenesis of glutamate 317 of bovine α-1,3Galactosyltransferase and its effect on enzyme activity: Implications for reaction mechanism

Bovine α1,3galactosyltransferase (α1,3GalT) transfers galactose from UDP-α-galactose to terminal β-linked galactosyl residues with retention of configuration of the incoming galactose residue. The epitope synthesized has been shown to be critical for xenotransplantation. According to a proposed double-displacement reaction mechanism, glutamate-317 (E317) is thought to be the catalytic nucleophile. The proposed catalytic role of E317 involves an initial nucleophilic attack with inversion of configuration and formation of a covalent sugar–enzyme intermediate between E317 and galactose from the donor substrate, followed by a second nucleophilic attack performed by the acceptor substrate with a second inversion of configuration. To determine whether E317 of α1,3GalT is critical for enzyme activity, site-directed mutagenesis was used to substitute alanine, aspartic acid, cysteine and histidine for E317. If the proposed reaction mechanism for the α1,3GalT enzyme is correct, E317D and E317H would produce active enzymes since they can act as nucleophiles. The non-conservative mutation E317A and conservative mutation E317C are predicted to produce inactive or very low activity enzymes since the E317A mutant cannot engage in a nucleophilic attack, and the E317C mutant would trap the galactose residue. The results obtained demonstrate that E317D and E317H mutants retained activity, albeit significantly less than the wild-type enzyme. Additionally, both E317A and E317C mutant also retained enzyme activity, suggesting that E317 is not the catalytic nucleophile proposed in the double-displacement mechanism. Therefore, either a different amino acid may act as the catalytic nucleophile or the reaction must proceed by a different mechanism.

Initiation of a Pre-Clinical Gene Therapy Study in Non-Human Primates Using Lentivectors Targeting Hematopoietic Cells for Correction of Fabry Disease.

Fabry disease is a lysosomal storage disorder (LSD) due to low or absent levels of α-galactosidase A (α-gal A). This results in accumulation of substrate with terminal galactosyl residues such as globotriaosylceramide (Gb3) in lysosomes causing pathology in different organs. Previously, we have demonstrated correction of the deficiency in Fabry mice in numerous gene therapy studies targeting hematopoietic cells. Here we have initiated a pre-clinical gene therapy study in non-human primates (NHPs) targeting Fabry disease. Three rhesus macaques are housed in our animal facility and implantation of telemetric devices and vascular access ports have occurred. We have mobilized hematopoietic stem/progenitor cells from all three animals independently by treatment for 5 days with 10 μg/kg/day of recombinant human granulocyte colony stimulating factor (rhuG-CSF) and 200 μg/kg/day recombinant human stem cell factor (rhuSCF). On the 5th day of mobilization, all animals underwent leukapheresis. To be more clinically relevant, we are using a protocol with an unmodified, commercially available apheresis machine for the rhesus macaques, which can be used for humans of an equivalent weight. From each successful apheresis, we collected approximately 1 x109/kg mobilized peripheral blood mononuclear cells (MoPBMNCs). After collection of MoPBMNCs, we isolated CD34+ cells using an anti-human CD34 antibody (clone 12.8) with a recovery of approximately 15–20 x 106 CD34+ cells per kg body weight of the animal with >80% purity. Collected CD34+ cells are stored in liquid nitrogen. These cells will be prestimulated for 24 hours with huSCF, huFlt3L, huIL-6 and huTPO (kindly provided by Amgen) and will be transduced with a concentrated bicistronic lentivector (LV) that engineers co-expression of huα-gal A and huCD25, a cell surface marker for transduced cells. Our lab has recently shown overexpression of a rhesus form of CD25 in >80% of transduced rhesus BM CD34+ cells mediated by a LV, validating its candidacy as a marker gene. Transduced cells will then be transplanted autologously in the NHPs after myeloablation by irradiation (10Gy) or mild chemotherapy (fludarabine and cyclophosphamide). The irradiation protocol has been optimized and a special plexiglass chamber, with the capacity for inhalational and intravenous anesthesia as well as a space for a HEPA filter, has been prepared for the animal procedures. The transplanted animals will be followed for at least one year and outcomes will be assessed by full measurement of safety parameters, α-gal A activity in plasma and relevant organs along with the real-time PCR and LAM PCR on BM and peripheral blood cells for the persistence of LV. Gb3 levels will also be examined in different organs compared to pre-transplant levels in tissue biopsies. We expect that this preclinical study in NHPs will serve as a roadmap to clinical gene therapy of Fabry disease using LV and provide important safety information for the use of this promising gene delivery system.

Enzymic transfer of α-L-arabinopyranosyl residues to exogenous 1,4-linked β-D-galacto-oligosaccharides using solubilized mung bean (Vigna radiata) hypocotyl microsomes and UDP-β-L-arabinopyranose

A single alpha-L-arabinopyranosyl (alpha-L-Arap) residue was shown, by a combination of chemical and spectroscopic methods, to be transferred to O-4 of the nonreducing terminal galactosyl (Gal) residue of 2-aminobenzamide (2AB)-labeled galacto-oligosaccharides when these oligosaccharides were reacted with UDP-ss-L-arabinopyranose (UDP-ss-L-Arap) in the presence of a Triton X-100-soluble extract of microsomal membranes isolated from mung bean (Vigna radiata, L. Wilezek) hypocotyls. Maximum-(1-->4)-arabinopyranosyltransferase activity was obtained at pH 6.0-6.5 and 20 degrees C in the presence of 25 mM Mn2+. The enzyme had an apparent K m of 45 microM for the 2AB-labeled galactoheptasaccharide and 330 microM for UDP-ss-L-Arap. A series of 2AB-labeled galacto-oligosaccharides with a degree of polymerization (DP) between 6 and 10 that contained a single alpha-L-Arap residue linked to the former nonreducing terminal Gal residue were generated when the 2AB-labeled galactohexasaccharide (Gal6-2AB) was reacted with UDP- ss-L-Ara p in the presence of UDP-beta-D-Galp and the solubilized microsomal fraction. The mono-arabinosylated galacto-oligosaccharides are not acceptor substrates for the galactosyltransferase activities known to be present in mung bean microsomes. These results show that mung bean hypocotyl microsomes contain an enzyme that catalyzes the transfer of Arap to the nonreducing Gal residue of galacto-oligosaccharides and suggest that the presence of a alpha-L-Arap residue on the former terminal Gal residue prevents galactosylation of galacto-oligosaccharides.

698. Gene Therapy of Murine GM1 Gangliosidosis by Genetically Modified Bone Marrow Hematopoietic Progenitor Cells

b-galactosidase (b-gal), a lysosomal enzyme involved the removal of b-linked terminal galactosyl residues of many glycoconjugates, is deficient the neurodegenerative lysosomal disorder GM1-gangliosidosis (GM1). GM1-/-mice closely mimic the most fundamental aspects of the neuropathological and neurochemical abnormalities of the human disorder. Bone marrow progenitor cells have been used as a source of corrective protein because of their ability to repopulate the recipients and to supply functional enzyme to different cells by in trans correction. Hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based bicistronic retroviral vector over-expressing b-gal and the green fluorescent protein (GFP) marker were used for transplantation into sublethally irradiated GM1-/-mice. Transduction efficiency of total BM cells with the MSCV-b-gal ranged from 25–89% prior to the transplantation. b-gal expressing BM-derived cells were detected histologically and enzymatically many tissues including spleen, liver, lungs, intestine and kidney after one, three and six months post transplantation (BMT). GFP-expressing cells of the erythroid, myeloid or lymphoid lineage were identified by FACS analysis of peripheral blood samples, collected at different time points after transplantation. Secondary transplantations demonstrated consistent long-term in vivo b-gal expression all tissues including brain. In addition, the thin layer chromatography of brain lipids showed a reduction the GM1-ganglioside content brainstem and cerebellum after three and six months post BMT. In line with the biochemical and histological findings, different behavioral tests including open field, rotorod and walking pattern, indicated a clear improvement of the neurological function transplanted GM1-/-compared to untreated littermates. Taken together these results have encouraged the use of ex-vivo gene therapy for the treatment of GM1.

Placental glycosylation in peccary species and its relation to that of swine and dromedary

Comparison has been made between glycans at the fetomaternal interface of two Tayassu species (New World peccaries or wild pigs) and those of swine (true pigs) and dromedary, which have similar epitheliochorial placentae. Plastic sections of near-term fetomaternal interface from Tayassu tajacu (120 days gestation) and Tayassu pecari (140 days gestation) were stained with 20 lectins and compared with those of swine (109 days) and dromedary (375 days). Both Tayassu species showed similar staining characteristics, which differed only slightly from those of the swine. Most differences were quantitative rather than qualitative, except for binding of Arachis hypogaea lectin to terminal β-galactose which was absent in swine uterine epithelium though present in both Tayassu species, and binding of Sambucus nigra lectin to sialic acid which was absent in swine epithelium and trophoblast though present in Tayassu. Glycosylation of the dromedary fetomaternal interface showed, in contrast, significant differences compared to Tayassu and swine, particularly regarding fucosyl, sialyl and terminal galactosyl residues. Despite a divergence of between 33 million and 37 million years between true pigs and peccaries, glycosylation of the fetomaternal interface has remained similar, with most of the observed changes affecting terminal structures. The dromedary has an epitheliochorial placenta with a similar architecture, but different glycan expression, suggesting modification of glycosyl transferases with evolution. These data contain clues to changes of glycosyl transferase activity that accompany speciation.