BBA - General Subjects (v.1573, #3)
Editorial Board (ii).
BBA special issue on developmental glycobiology by Scott B Selleck; Harry Schachter (199).
Johannes Frederik Gerardus Vliegenthart by Hans Kamerling (201-205).
Thanks to Mr. Sialic Acid by Sörge Kelm; Anthony Corfield (207-208).
Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes by Kay Grobe; Johan Ledin; Maria Ringvall; Katarina Holmborn; Erik Forsberg; Jeffrey D Esko; Lena Kjellén (209-215).
Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.
Keywords: Heparan sulfate; Mast cell; Heparin; Embryonic development; Translational regulation; Gene targeting;
Mutation of Large, which encodes a putative glycosyltransferase, in an animal model of muscular dystrophy by Prabhjit K Grewal; Jane E Hewitt (216-224).
The myodystrophy (myd) mutation arose spontaneously and has an autosomal recessive mode of inheritance. Homozygous mutant mice display a severe, progressive muscular dystrophy. Using a positional cloning approach, we identified the causative mutation in myd as a deletion within the Large gene, which encodes a putative glycosyltransferase with two predicted catalytic domains. By immunoblotting, the α-subunit of dystroglycan, a key muscle membrane protein, is abnormal in myd mice. This aberrant protein might represent altered glycosylation of the protein and contribute to the muscular dystrophy phenotype. Our results are discussed in the light of recent reports describing mutations in other glycosyltransferase genes in several forms of human muscular dystrophy.
Keywords: Carbohydrate; Glycosyltransferase; Muscular dystrophy; Mouse mutant; Dystroglycan;
Golgi α-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals by Kelley W Moremen (225-235).
The maturation of N-glycans to complex type structures on cellular and secreted proteins is essential for the roles that these structures play in cell adhesion and recognition events in metazoan organisms. Critical steps in the biosynthetic pathway leading from high mannose to complex structures include the trimming of mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and Golgi complex. These exo-mannosidases comprise two separate families of enzymes that are distinguished by enzymatic characteristics and sequence similarity. Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include enzymes involved in trimming reactions in N-glycan maturation in the Golgi complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and cytosol. Studies on the biological roles of complex type N-glycans have employed a variety of strategies including the treatment of cells with glycosidase inhibitors, characterization of human patients with enzymatic defects in processing enzymes, and generation of mouse models for the enzyme deficiency by selective gene disruption approaches. Corresponding studies on Golgi mannosidase II have employed swainsonine, an alkaloid natural plant product that causes “locoism”, a phenocopy of the lysosomal storage disease, α-mannosidosis, as a result of the additional targeting of the broad-specificity lysosomal mannosidase by this compound. The human deficiency in Golgi mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Mouse models for Golgi mannosidase II deficiency recapitulate many of the pathological features of the human disease and confirm that the unexpectedly mild effects of the enzyme deficiency result from a tissue-specific and glycoprotein substrate-specific alternate pathway for synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of a systemic autoimmune disorder as a consequence of the altered glycosylation. This review will discuss the biochemical features of Golgi mannosidase II and the consequences of its deficiency in mammalian systems as a model for the effects of alterations in vertebrate N-glycan maturation during development.
Keywords: Golgi mannosidase II; Oligosaccharide; Mammal;
Early developmental expression of the gene encoding glucosylceramide synthase, the enzyme controlling the first committed step of glycosphingolipid synthesis by Tadashi Yamashita; Ryuichi Wada; Richard L Proia (236-240).
Glycosphingolipids (GSLs) are ubiquitous plasma membrane components composed of a ceramide lipid anchor attached to one of a diverse complement of oligosaccharide structures. Fundamentally important activities have been attributed to GSLs including formation of plasma membrane structures involved in membrane trafficking, signal transduction and cell–cell interactions. Glucosylceramide synthase converts ceramide to glucosylceramide, a core structure of the vast majority of GSLs. Disruption of the gene encoding glucosylceramide synthase (Ugcg) caused embryonic lethality in mice during gastrulation. To further investigate the role of GSL synthesis during embryogenesis, we produced mice with a Lacz reporter gene inserted into the glucosylceramide synthase locus. These mice allowed the visualization of glucosylceramide synthase expression during early embryonic development.
Keywords: Glucosylceramide synthase; Glycosphingolipid; UDP-glucose;
The role of glypicans in mammalian development by Howard H Song; Jorge Filmus (241-246).
Glypicans are a family of heparan sulfate proteoglycans that are bound to the cell surface by a glycosyl-phosphatidylinositol anchor. Six members of this family have been identified in mammals. In general, glypicans are highly expressed during development, and their expression pattern suggests that they are involved in morphogenesis. One member of this family, glypican-3, is mutated in the Simpson–Golabi–Behmel syndrome. This syndrome is characterized by overgrowth and various developmental abnormalities that indicate that glypican-3 inhibits proliferation and cell survival in the embryo. It has consequently been proposed that glypicans can regulate the activity of several growth factors that play a critical role in morphogenesis.
Keywords: Proteoglycan; Heparan sulfate; Glypican; Mammalian development;
The Caenorhabditis elegans sqv genes and functions of proteoglycans in development by Dorota A Bulik; Phillips W Robbins (247-257).
In the nematode Caenorhabditis elegans, the vulva is a simple tubular structure linking the gonads with the external cuticle. In this review we summarize knowledge of inter- and intracellular signaling during vulval development and of the genes required for vulval invagination. Mutants of one set of these genes, the sqv genes, have a normal number of vulval precursor cells (VPCs) with an unperturbed cell lineage but the invagination space, normally a tube, is either collapsed or absent. We review evidence that the sqv genes are involved in glycosaminoglycan synthesis and speculate on ways in which defective glycosaminoglycan formation might lead to collapse of the vulval structure.
Keywords: Caenorhabditis elegans; sqv gene; Proteoglycan; Glycosaminoglycan; Vulva;
Targeted mutations in β1,4-galactosyltransferase I reveal its multiple cellular functions by Carey Rodeheffer; Barry D Shur (258-270).
β1,4-Galactosyltransferase I (GalT I) is one of the most extensively studied glycosyltransferases. It is localized in the trans-Golgi compartment of most eukaryotic cells, where it participates in the elongation of oligosaccharide chains on glycoproteins and glycolipids. GalT I has also been reported in non-Golgi locations, most notably the cell surface, where it has been suggested to function non-biosynthetically as a receptor for extracellular glycoside substrates. Cloning of the GalT I cDNAs revealed that the gene encodes two similar proteins that differ only in the length of their cytoplasmic domains. Whether these different GalT I proteins, or isoforms, have similar or different biological roles is a matter of active investigation. The functions of the GalT I proteins have been addressed by targeted mutations that eliminate either both GalT I isoforms or just the long GalT I isoform. Eliminating both GalT I proteins abolishes most, but not all, GalT activity, an observation that led to the realization that other GalT family members must exist. The loss of both GalT I isoforms leads to neonatal lethality due to a wide range of phenotypic abnormalities that are most likely the result of decreased galactosylation. When the long isoform of GalT I is eliminated, galactosylation proceeds grossly normal via the short GalT I isoform, but specific defects in cell interactions occur that are thought to depend upon a non-biosynthetic function of the long GalT I isoform.
Keywords: β1,4-Galactosyltransferase I; Glycosylation; Golgi complex; Plasma membrane; Fertilization; Mammary gland;
UDP-N-acetylglucosamine:α-3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:α-6-d-mannoside β-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans by Shihao Chen; Jenny Tan; Vernon N Reinhold; Andrew M Spence; Harry Schachter (271-279).
UDP-N-acetylglucosamine:α-3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:α-6-d-mannoside β-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.
Keywords: Complex N-glycans; Null mutation; UV-TMP mutagenesis; Worm development;
Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila by Kent Nybakken; Norbert Perrimon (280-291).
Heparan sulphate proteoglycans (HSPG's) are cell surface proteins to which long, unbranched chains of modified sugars called heparan sulphate glycosaminoglycans have been covalently attached. Cell culture studies have demonstrated that HSPG's are required for optimal signal transduction by many secreted cell signaling molecules. Now, genetic studies in both Drosophila and vertebrates have illustrated that HSPG's play important roles in signal transduction in vivo and have also begun to reveal new roles for HSPG's in signaling events. In particular, HSPG's have been shown to be important in ligand sequestration of wingless, for the transport of the Hedgehog ligand, and for modulation of the Dpp morphogenetic gradient.
Keywords: Heparan sulfate; Developmental signaling; Drosophila; Hedgehog signaling; FGF signaling; Dpp signaling;
The role of the GlcNAcβ1,2Manα- moiety in mammalian development. Null mutations of the genes encoding UDP-N-acetylglucosamine:α-3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:α-d-mannoside β-1,2-N-acetylglucosaminyltransferase I.2 cause embryonic lethality and congenital muscular dystrophy in mice and men, respectively by Harry Schachter (292-300).
The GlcNAcβ1,2Manα- moiety can be synthesized by at least two mammalian glycosyltransferases, UDP-GlcNAc:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I, EC 220.127.116.11) and UDP-GlcNAc:α-d-mannoside β1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). GnT I adds a GlcNAc residue in β1,2 glycosidic linkage to the Manα1,3 arm of the N-glycan core to initiate the biosynthesis of hybrid and complex N-glycans. GnT I.2 can add GlcNAc in β1,2 linkage to any α-linked terminal Man residue but has a strong preference for the Manα1-O-Thr- moiety which occurs in α-dystroglycan and other O-mannosylated glycoproteins. Mouse embryos lacking a functional GnT I gene (MgatI) were unable to synthesize complex N-glycans and none survived past 10.5 days after fertilization. The embryos showed multisystemic defects in various morphogenic processes such as neural tube formation, vascularization and the determination of left–right body plan asymmetry. Six human patients with muscle-eye-brain disease (MEB) were recently shown to have point mutations in the gene encoding GnT I.2 (MGATI.2). MEB is an autosomal recessive disease characterized by congenital muscular dystrophy, ocular abnormalities, brain malformations and other multisystemic defects. Both the MGATI.2 gene and MEB disease have been mapped to chromosome 1p32–p34. At least one of the biochemical sites affected by the MGATI.2 mutations is probably the interaction between laminin in the extracellular matrix and the peripheral membrane glycoprotein α-dystroglycan since this interaction is believed to require the presence of the sialylα2,3Galβ1,4GlcNAcβ1,2Manα1-O-Ser/Thr moiety on α-dystroglycan. It can be concluded that the GlcNAcβ1,2Manα- moiety is important for mammalian development due to an essential role in two distinct biochemical pathways.
Keywords: Complex N-linked glycans; Gene targeting; Mammalian ontogeny; Congenital muscular dystrophy; O-mannosylation;
Mice with a homozygous deletion of the Mgat2 gene encoding UDP-N-acetylglucosamine:α-6-d-mannoside β1,2-N-acetylglucosaminyltransferase II: a model for congenital disorder of glycosylation type IIa by Yan Wang; Harry Schachter; Jamey D Marth (301-311).
Mice homozygous for a deletion of the Mgat2 gene encoding UDP-N-acetylglucosamine:α-6-d-mannoside β1,2-N-acetylglucosaminyltransferase II (GlcNAcT-II, EC 18.104.22.168) have been reported. GlcNAcT-II is essential for the synthesis of complex N-glycans. The Mgat2-null mice were studied in a comparison with the symptoms of congenital disorder of glycosylation type IIa (CDG-IIa) in humans. Mutant mouse tissues were shown to be deficient in GlcNAcT-II enzyme activity and complex N-glycan synthesis, resulting in severe gastrointestinal, hematologic and osteogenic abnormalities. All mutant mice died in early post-natal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors exhibiting additional and novel disease signs of CDG-IIa. Analysis of N-glycan structures in the kidneys of Mgat2-null mice showed a novel bisected hybrid N-glycan structure in which the bisecting GlcNAc residue was substituted with a β1,4-linked galactose or the Lewis x structure. These studies suggest that some of the functions of complex N-glycan branches are conserved in mammals and that human disease due to aberrant protein N-glycosylation may be modeled in the mouse, with the expectation in this case of gaining insights into CDG-IIa disease pathogenesis. Further analyses of the Mgat2-deficient phenotype in the mouse have been accomplished involving cells in which the Mgat2 gene is dispensable, as well as other cell lineages in which a severe defect is present. Pre-natal defects appear in a significant number of embryos, and likely reflect a limited window of time in which a future therapeutic approach might effectively operate.
Keywords: Genetics; Disease; N-glycan; Glycosylation; CDG-IIa;
Heparan sulfate fine structure and specificity of proteoglycan functions by Hiroshi Nakato; Koji Kimata (312-318).
Heparan sulfate chains have markedly heterogeneous structures in which distinct patterns of sulfation determine the binding specificity for ligand proteins. These “fine structures” of heparan sulfate are mainly produced by the regulated introduction of sulfate groups at the N-, 2-O-, 6-O-, and 3-O-positions of the sugar chain. Recent biochemical, histochemical, and genetic studies have demonstrated that different fine structures mediate distinct molecular recognition events to regulate a variety of cellular functions. In this review, we focus on the molecular basis of growth factor control by the sulfation status of heparan sulfate.
Keywords: Proteoglycan; Heparan sulfate; Fine structure; Sulfotransferase; Sulfatase; Tracheal system; Drosophila;
Role of heparan sulfate-2-O-sulfotransferase in the mouse by Catherine L.R Merry; Valerie A Wilson (319-327).
Heparan sulfate (HS) is a long unbranched polysaccharide found covalently attached to various proteins at the cell surface and in the extracellular matrix. It plays a central role in embryonic development and cellular function by modulating the activities of an extensive range of growth factors and morphogens. HS 2-O-sulfotransferase (Hs2st) occupies a critical position in the succession of enzymes responsible for the biosynthesis of HS, catalysing the transfer of sulfate to the C2-position of selected hexuronic acid residues within the nascent HS chain. Previous studies have concluded that 2-O-sulfation of HS is essential for it to cooperate in many growth factor/receptor interactions. Surprisingly therefore, embryos lacking functional Hs2st survive until birth, but die perinatally, suffering complete failure to form kidneys. However, this rather late lethality belies a more intricate involvement of 2-O-sulfated HS during development. The purpose of this review is to summarise the requirements for 2-O-sulfated HS during mouse development, at the morphological and molecular level. The implications that altered HS structure may have on growth factor/receptor signalling in vivo will be discussed.
Keywords: Heparan sulfate; Heparan sulfate 2-O-sulfotransferase; Hs2st; Mouse; Embryo;
Modulation of receptor signaling by glycosylation: fringe is an O-fucose-β1,3-N-acetylglucosaminyltransferase by Robert S. Haltiwanger; Pamela Stanley (328-335).
The Notch family of signaling receptors plays key roles in determining cell fate and growth control. Recently, a number of laboratories have shown that O-fucose glycans on the epidermal growth factor (EGF)-like repeats of the Notch extracellular domain modulate Notch signaling. Fringe, a known modifier of Notch function, is an O-fucose specific β1,3-N-acetylglucosaminyltransferase. The transfer of GlcNAc to O-fucose on Notch by fringe results in the potentiation of signaling by the Delta class of Notch ligands, but causes inhibition of signaling by the Serrate/Jagged class of Notch ligands. Interestingly, addition of a β1,4 galactose by β4GalT-1 to the GlcNAc added by fringe is required for Jagged1-induced Notch signaling to be inhibited in a co-culture assay. Thus, both fringe and β4GalT-1 are modulators of Notch function. Several models have been proposed to explain how alterations in O-fucose glycans result in changes in Notch signaling, and these models are discussed.
Keywords: Fringe; Notch; O-fucose; Signal transduction; N-acetylglucosaminyltransferase;
Intracellular glycosylation and development by Niall O'Donnell (336-345).
O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification of cytoplasmic and nuclear proteins. Although the function of this abundant modification is yet to be definitively elucidated, all O-GlcNAc proteins are phosphoproteins. Further, the serine and threonine residues substituted with O-GlcNAc are often sites of, or close to sites of, protein phosphorylation. This implies that there may be a dynamic interplay between these two post-translational modifications to regulate protein function. In this review, the functions of some of the proteins that are modified by O-GlcNAc will be considered in the context of the potential role of the O-GlcNAc modification. Furthermore, predictions will be made as to how cellular function and developmental regulation might be affected by changes in O-GlcNAc levels.
Keywords: O-GlcNAc; O-GlcNAc transferase; UDP-GlcNAc metabolism; Transcription factor; Protein degradation;
Hereditary multiple exostoses and heparan sulfate polymerization by Beverly M Zak; Brett E Crawford; Jeffrey D Esko (346-355).
Hereditary multiple exostoses (HME, OMIM 133700, 133701) results from mutations in EXT1 and EXT2, genes encoding the copolymerase responsible for heparan sulfate (HS) biosynthesis. Members of this multigene family share the ability to transfer N-acetylglucosamine to a variety of oligosaccharide acceptors. EXT1 and EXT2 encode the copolymerase, whereas the roles of the other EXT family members (EXTL1, L2, and L3) are less clearly defined. Here, we provide an overview of HME, the EXT family of proteins, and possible models for the relationship of altered HS biosynthesis to the ectopic bone growth characteristic of the disease.
Keywords: Chondrocyte; Chondrosarcoma; Bone development; Biosynthesis; EXT;
β1,4-N-acetylgalactosaminyltransferase—GM2/GD2 synthase: a key enzyme to control the synthesis of brain-enriched complex gangliosides by Koichi Furukawa; Kogo Takamiya; Keiko Furukawa (356-362).
β1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) is a key enzyme which catalyzes the conversion of GM3, GD3 and lactosylceramide (LacCer) to GM2, GD2 and asialo-GM2 (GA2), respectively. This step is critical for the synthesis of all complex gangliosides enriched in the nervous system of vertebrates. Following the cloning of cDNAs encoding GM2/GD2 synthase by an expression cloning approach, substantial evidence for the roles of complex gangliosides have been obtained. Above all, knock-out mice lacking all complex gangliosides revealed important roles of complex gangliosides in vivo, i.e., in the maintenance and repair of nervous tissues, in the intact differentiation of spermatocytes via the transport of testosterone, and in the regulation of interleukin-2 receptor complex. Molecular mechanisms for these functions of complex gangliosides in vivo remain to be clarified.
Keywords: GM2; GD2; Ganglioside; GM2/GD2 synthase; Knock-out; Targeting;
Biological consequences of overexpressing or eliminating N-acetylglucosaminyltransferase-TIII in the mouse by Pamela Stanley (363-368).
N-acetylglucosaminyltransferase III (GlcNAc-TIII), a product of the human MGAT3 gene, was discovered as a glycosyltransferase activity in hen oviduct. GlcNAc-TIII transfers GlcNAc in β4-linkage to the core Man of complex or hybrid N-glycans, and thereby alters not only the composition, but also the conformation of the N-glycan. The dramatic consequences of the addition of this bisecting GlcNAc residue are reflected in the altered binding of lectins that recognize Gal residues on N-glycans. Changes in GlcNAc-TIII expression correlate with hepatoma and leukemia in rodents and humans, and the bisecting GlcNAc on Asn 297 of human IgG antibodies enhances their effector functions. Overexpression of a cDNA encoding GlcNAc-TIII alters growth control and cell–cell interactions in cultured cells, and in transgenic mice. While mice lacking GlcNAc-TIII are viable and fertile, they exhibit retarded progression of diethylnitrosamine (DEN)-induced liver tumors. Further biological functions of GlcNAc-TIII are expected to be uncovered as mice with a null mutation in the Mgat3 gene are challenged.
Keywords: N-acetylglucosaminyltransferase; GlcNAc-TIII; Bisecting GlcNAc; Tumor progression; Growth control;
Glycobiology of the synapse: the role of glycans in the formation, maturation, and modulation of synapses by Yu Yamaguchi (369-376).
Synapses, which are the fundamental functional unit of the nervous system, are considered to be highly specialized cell adhesion structures. Studies since the 1960s demonstrated that various carbohydrates and glycoproteins are expressed in synapses in the central and peripheral nervous system. Although the functional roles of these synaptic carbohydrates and glycoproteins remain to be determined, rapidly accumulating data suggest that they may play critical roles in the formation, maturation, and functional modulation of synapses.
Keywords: Synapse; Neuromuscular junction; Polysialic acid; Heparan sulfate; Syndecan-2; Agrin;
Galactosyltransferase I is a gene responsible for progeroid variant of Ehlers-Danlos syndrome: molecular cloning and identification of mutations by Koichi Furukawa; Tetsuya Okajima (377-381).
A human cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated, showing a type II membrane protein with 327 amino acids and 38% homology to the Caenorhabditis elegans sqv-3 gene involved in vulval invagination and oocyte development. This cDNA exhibited marked galactosyltransferase activity specific for p-nitrophenyl-β-d-xylopyranoside, and also restored glycosaminoglycan (GAG) synthesis to galactosyltransferase I-deficient CHO mutant pgsB-761 cells. The enzyme product contained β-1,4-linked galactosyl residues, indicating that the enzyme is galactosyltransferase I (UDP-d-galactose: d-xylose β-1,4-d-galactosyltransferase; EC 22.214.171.124) involved in the synthesis of the GAG–protein linkage region of proteoglycans. Mutations of this gene were investigated in a case of Ehlers-Danlos syndrome (progeroid variant), since reduced activity of galactosyltransferase I had been reported in this disease by others. As expected, the patient gene contained two different mutations (A186D, L206P). The mutations showed, respectively, 10–50% and 0% of the enzyme activity compared with wild type, suggesting that galactosytransferase I (XGal-T1) is at least one of the genes responsible for Ehlers-Danlos syndrome (progeroid variant).
Keywords: Galactosyltransferase I; Ehlers-Danlos syndrome; Mutation;
In vivo role of α-mannosidase IIx: ineffective spermatogenesis resulting from targeted disruption of the Man2a2 in the mouse by Michiko N Fukuda; Tomoya O Akama (382-387).
Alpha-mannosidase IIx (MX) is an enzyme closely related to the Golgi N-glycan processing enzyme α-mannosidase II (MII). The enzymatic activity of MX in vitro is minimal. Therefore, the in vivo role of MX in N-glycan processing is as yet unclear. The targeted disruption of the gene encoding MX in the mouse resulted in an obvious phenotype, i.e., MX-deficient males were found to be infertile. Testes from homozygous mutant male mice are smaller than those from wild-type or heterozygous littermates. Histology of the MX null mouse testis showed significant reduction of spermatogenic cells in the seminiferous tubules. Electron microscopy showed that prominent intercellular spaces surround MX-deficient spermatogenic cells, suggesting a failure of germ cell adhesion to Sertoli cells. Quantitative structural analyses of N-glycans from wild-type and MX-deficient mouse testis showed that wild-type testes contain GlcNAc-terminated complex type N-glycans, while they are significantly reduced in MX-deficient mutant testis. An in vitro assay for adhesion of spermatogenic cells to Sertoli cells was carried out. By testing the effect of each purified N-glycan oligosaccharide, it was demonstrated that a GlcNAc-terminated tri-antennary, fucosylated N-glycan has an activity on the adhesion between germ cells and Sertoli cells. Thus, the targeted disruption of the gene encoding MX uncovered a novel carbohydrate recognition system in a biologically important process, spermatogenesis.
Keywords: N-glycan processing; Mannosidase; Infertility; Adhesion; Sertoli cell;
Human disorders in N-glycosylation and animal models by Hudson H Freeze (388-393).
Genes that cause human disorders in N-linked oligosaccharide biosynthesis have appeared much faster than animal model systems to study them. In most models, a single gene is altered or deleted while other genes and the environment are held constant. Since humans have variable genetic backgrounds and environments, model systems may only partially mimic the actual disorders. Mutations in seven of the 30–40 genes needed for the synthesis and transfer of oligosaccharides from the lipid donor to the nascent protein acceptors in the endoplasmic reticulum cause Type I Congenital Disorders of Glycosylation (CDG). Since all of these gene products ultimately contribute to the same final step, one might suspect that all the diseases would be very similar. However, even patients with mutations in the same gene show considerable phenotypic variability. Modifier, or susceptibility genes in the background likely explain some variations of the “primary” gene chosen for study. Add to this the stress of infections, dietary insufficiencies, and the demands of growth itself. These issues are particularly important during development when the temporal and spatial specific interplay of cell adhesions and signals has only a single opportunity. Multiple hypomorphic alleles of genes in the same pathway may have synergistic effects. Investigators designing model systems to study human glycosylation disorders may want to construct strains with several heterozygous hypomorphic alleles in rate-limiting steps in the glycosylation pathway.
Keywords: Congenital Disorder of Glycosylation; Animal model; N-glycosylation; Human disease;
Roles of mucin-type O-glycans in cell adhesion by Minoru Fukuda (394-405).
Mucin-type O-glycans containing Core2 branches have distinctly different functions from those O-glycans that contain Core1 structures. Core2 branched O-glycans can have terminal structures that function as ligands for carbohydrate binding proteins. However, sialylated Core2 branched O-glycans without additional modifications exhibit anti-adhesive properties. These results demonstrate that certain mucin-type O-glycans can either facilitate or attenuate cell adhesion depending on the core structures and the structures of the non-reducing termini.
Keywords: Mucin-type O-glycan; Core2 branching β1,6-N-acetylglucosaminyltransferase; Protein–protein interaction; Carbohydrate–protein interaction;
Galactolipids are molecular determinants of myelin development and axo–glial organization by Jill Marcus; Brian Popko (406-413).
Myelination is a developmentally regulated process whereby myelinating glial cells elaborate large quantities of a specialized plasma membrane that ensheaths axons. The myelin sheath contains an unusual lipid composition in that the glycolipid galactosylceramide (GalC) and its sulfated form sulfatide constitute a large proportion of the total lipid mass. These glycolipids have been implicated in a range of developmental processes such as cell differentiation and myelination initiation, but analyses of mice lacking UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme required for myelin galactolipid synthesis, have more recently demonstrated that the galactolipids more subtly regulate myelin formation. The CGT mutants display a delay in myelin maturation and axo–glial interactions develop abnormally. By interbreeding the CGT mutants with mice that lack myelin-associated glycoprotein, it has been shown that these specialized myelin lipids and proteins act in concert to promote axo–glial adhesion during myelinogenesis. The analysis of the CGT mutants is helping to clarify the roles myelin galactolipids play in regulating the development, and ultimately the function of the myelin sheath.
Keywords: Galactolipid; Myelination; Axo–glial interaction; Node;
UDP-N-acetylglucosamine:α-6-d-mannoside β1,6 N-acetylglucosaminyltransferase V (Mgat5) deficient mice by James W. Dennis; Judy Pawling; Pam Cheung; Emily Partridge; Michael Demetriou (414-422).
Targeted gene mutations in mice that cause deficiencies in protein glycosylation have revealed functions for specific glycans structures in embryogenesis, immune cell regulation, fertility and cancer progression. UDP-N-acetylglucosamine:α-6-d-mannoside β1,6 N-acetylglucosaminyltransferase V (GlcNAc-TV or Mgat5) produces N-glycan intermediates that are elongated with poly N-acetyllactosamine to create ligands for the galectin family of mammalian lectins. We generated Mgat5-deficient mice by gene targeting methods in embryonic stem cells, and observed a complex phenotype in adult mice including susceptibility to autoimmune disease, reduced cancer progression and a behavioral defect. We found that Mgat5-modified N-glycans on the T cell receptor (TCR) complex bind to galectin-3, sequestering TCR within a multivalent galectin–glycoprotein lattice that impedes antigen-dependent receptor clustering and signal transduction. Integrin receptor clustering and cell motility are also sensitive to changes in Mgat5-dependent N-glycosylation. These studies demonstrate that low affinity but high avidity interactions between N-glycans and galectins can regulate the distribution of cell surface receptors and their responsiveness to agonists.
Keywords: N-glycan; Cancer; Immunity; Mgat5; Poly N-acetyllactosamine;
General Subjects Author Index (423-425).
General Subjects Cumulative Contents (426-428).