Conformational and electrostatic analysis of SN1 donor analogue glycomimetic inhibitors of ST3Gal-I mammalian sialyltransferase

Mammalian sialyltransferases play a role in the metastasis of various cancers in humans. Inhibitors of these enzymes will in principle be able to directly inhibit aberrant sialylation in cancer. Inhibitors of ST3Gal-I resembling the donor component of S_N1 Transition State structures were previously evaluated as part of a kinetics study. Here, using classical dynamics simulations and free energy perturbation calculations, we rationalize the performance of three of these donor analogue ST3Gal-I enzyme inhibitors. We find to inhibit the mammalian ST3Gal-I enzyme a donor analogue requires configurationally limited functionality. This is mediated by the binding of the inhibitor to the enzyme. The inhibitor’s ability to interact with Y194 and T272 through a charged group such as a carboxylate is especially important. Furthermore, a conformational rigid form approximating the donor substrate is central. Here this is achieved by an intramolecular hydrogen bond formed between the carboxylate group and one of the ribose hydroxyl groups of the cytidine monophosphate (CMP) leaving group. This intramolecular interaction results in the donor substrate conformer complimenting the form of the catalytic binding site. Finally the carboxylate charge is essential for electrostatic pairing with the binding site. Substituting this group for an alcohol or amide results in severe weakening of the ligand binding. The carboxylate thus proves an to be an irreplaceable functional group and an essential pharmacophore.     

ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling

The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation.     

Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.     

Proteolytic shedding of ST6Gal-I by BACE1 regulates the glycosylation and function of alpha4beta1 integrins

Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due in part to the activation of alpha4beta1 integrins. Here we report that the sustained alpha4beta1 activation associated with macrophage differentiation results from expression of beta1 integrin subunits that lack alpha2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14(+) monocytes, ST6Gal-I is down-regulated, leading to beta1 hyposialylation and enhanced alpha4beta1-dependent VCAM-1 binding. Importantly, ST6Gal-I down-regulation results from cleavage by the BACE1 secretase, which we show is dramatically up-regulated during macrophage differentiation. BACE1 up-regulation, ST6Gal-I shedding, beta1 hyposialylation, and alpha4beta1-dependent VCAM-1 binding are all temporally correlated and share the same signaling mechanism (protein kinase C/Ras/ERK). Preventing ST6Gal-I down-regulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the beta1 subunit. Collectively, these results describe a novel mechanism for alpha4beta1 regulation and further suggest an unanticipated role for BACE1 in macrophage function.     

Genetic linkage analysis of X-ray hypersensitivity in the LEC mutant rat

The LEC rat has been reported to exhibit X-ray hypersensitivity and deficiency in DNA double-strand break (DSB) repair. The present study was performed to map the locus responsible for this phenotype, the xhs (X-ray hypersensitivity), as the first step in identifying the responsible gene. Analysis of the progeny of (BN × LEC)F₁× LEC backcrosses indicated that the X-ray hypersensitive phenotype was controlled by multiple genetic loci in contrast to the results reported previously. Quantitative trait loci (QTL) linkage analysis revealed two responsible loci located on Chromosomes (Chr) 4 and 1. QTL on Chr 4 exhibited very strong linkage to the X-ray hypersensitive phenotype, while QTL on Chr 1 showed weak linkage. The Rad52 locus, mutation of which results in hypersensitivity to ionizing radiation and impairment of DNA DSB repair in yeast, was reported to be located on the synteneic regions of mouse Chr 6 and human Chr 12. However, mapping of the rat Rad52 locus indicated that it was located 23 cM distal to the QTL on Chr 4. Furthermore, none of the radio-sensitivity-related loci mapped previously in the rat chromosome were identical to the QTL on Chrs 4 and 1 in the LEC rat. Thus, it seems that X-ray hypersensitivity in the LEC rat is caused by mutation(s) in as-yet-undefined genes. Received: 14 February 2000 / Accepted: 17 May 2000     

Molecular genetic linkage maps of mouse chromosomes 4 and 6.

We have generated a moderate resolution genetic map of mouse chromosomes 4 and 6 utilizing a (C57BL/6J x Mus spretus) F1 x Mus spretus backcross with RFLPs for 31 probes. The map for chromosome 4 covers 77 cM and details a large region of homology to human chromosome 1p. The map establishes the breakpoints in the mouse 4-human 1p region of homology to a 2-cM interval between Ifa and Jun in mouse and to the interval between JUN and ACADM in human. The map for mouse chromosome 6 spans a 65-cM region and contains a large region of homology to human 7q. These maps also provide chromosomal assignment and order for a number of previously unmapped probes. The maps should allow the rapid regional assignment of new markers to mouse chromosomes 4 and 6. In addition, knowledge of the gene order in mouse may prove useful in determining the gene order of the homologous regions in human.     

Molecular analysis of an acatalasemic mouse mutant

The Csb acatalasemia mouse mutant differentially expresses reduced levels of catalase activity in a tissue specific manner. In order to pinpoint the molecular lesion that imparts the acatalasemia phenotype in Csb mice we have utilized the polymerase chain reaction technique to isolate catalase cDNA clones from control and Csb mouse strains. Sequence analyses of these cDNA clones have revealed a single nucleotide difference within the coding region of catalase between control and Csb mice. This nucleotide transversion (G----T) is located in the third position of amino acid 11 in the catalase monomer. In control mouse strains glutamine (CAG) is encoded at amino acid 11, while in Csb mice this codon (CAT) encodes histidine. This amino acid is located within a region that forms the first major alpha-helix in the amino-terminal arm of the catalase subunit and, as such, may render the catalase molecule unstable under certain physiological conditions.     

Cellular analysis of limb development in the mouse mutant Hypodactyly

The limb defect in the mouse Hypodoctyly (Hd) affects only the distal structures. Heterozygotes (Hd/+) lack all or part of the distal phalanx and the terminal claw of digit 1 on the hindlimbs; mice homozygous (Hd/Hd) for the mutation have just one digit on each of the four limbs. Early limb development in the mutant appears normal and a change in morphology can only be detected later. Limb buds of Hd/+ and Hd/Hd embryos become reduced in width, with Hd/Hd buds becoming very pointed instead of rounded. This change in bud shape is correlated with an increase in cell death anteriorly in Hd/+ hindlimbs and both anteriorly and posteriorly in Hd/Hd fore- and hindlimb buds. The apical ectodermal ridge is very pronounced in pointed Hd/Hd limb buds. Mesenchyme cells from the Hd/Hd mutant in culture show a cell-autonomous change in behaviour and less cartilage differentiates. © 1996 Wiley-Liss, Inc.     

Immunofluorescent analysis of fibronectin and laminin distribution in the vl mutant mouse

Summary The distribution of fibronectin and laminin was determined in the basement membrane surrounding the caudal neural tube and at the site of initial apposition of the caudal neural folds by means of indirect immunofluorescence histochemistry on 9.0- to 10.5-day mouse embryos fixed in Carnoy's solution and serially sectioned in paraffin. At early phases of development of normal (+/+) and abnormal (vl/vl) embryos the dorsolateral neural basement membrane overlying putative neural crest cells caudal to the hindlimb shows a patchy fibronectin reaction, with laminin virtually absent. In older embryos, both components are present but are discontinuous overlying the neural crest. The results suggest that since discontinuities occur in the basement membrane of abnormal as well as normal embryos, the neural crest cells are not prevented from emigrating from the abnormal neural tube; thus the faulty neural fold fusion that characterizesvl/vl embryos does not appear to be due to a suppression of emigration by the basement membrane. The results also demonstrate the advantages and reliability of embedding in paraffin for analysis of serially sectioned pathological material by means of indirect immunofluorescence, provided that normal controls and abnormals are processed simultaneously.     

Isolation and analysis by the reductive-cleavage method of linkage positions and ring forms in the Mycobacterium smegmatis cell-wall arabinogalactan

The Mycobacterium smegmatis arabinogalactan polysaccharide has been isolated from the cell wall by saponification and extraction to remove lipids and subsequent solubilization by treatment with lysozyme. Analysis for neutral sugars demonstrated the presence of D-arabinose and D-galactose in a ratio of 3:1, respectively. Reductive cleavage of the fully methylated polysaccharide in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate and subsequent acetylation in situ gave six partially methylated 1,4-anhydroalditol acetates as the major products and three partially methylated 1,5-anhydroalditol acetates as minor products. Partially methylated 1,5-anhydroalditol acetates were not formed when reductive cleavage was accomplished with triethylsilane and a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate as the catalyst, demonstrating that the polysaccharide is exclusively comprised of furanosyl residues. The partially methylated anhydroalditols so produced were identified by comparison to authentic standards. Their identifies are consistent with the presence in the M. smegmatis arabinogalactan of an octasaccharide repeating unit comprised of a nonreducing terminal D-arabinofuranosyl group, a 2-O-linked D-arabinofuranosyl residue, three 5-O-linked D-arabinofuranosyl residues, a 3,5-di-O-linked D-arabinofuranosyl residue, a 5-O-linked D-galactofuranosyl residue, and a 6-O-linked D-galactofuranosyl residue.     

Analysis of linkage positions in saccharomyces cerevisiaed-mannans by the reductive-cleavage method

The positions of linkage in the d-mannans derived from Saccharomyces cerevisiae X2180 and its mutants, mnn1, mnn2, and mnn4, were established by perethylation and subsequent reductive cleavage with triethylsilane in the presence of boron trifluoride etherate (BF₃ · Et₂O) or trimethylsilyl trifluoromethanesulfonate. With the latter as the catalyst, all glycosidic carbon-oxygen bonds were cleaved, to produce a mixture of ethylated 1,5-anhydro-d-mannitol derivatives. With BF₃ · Et₂O as the catalyst, 2-, 3-, and 6-linked residues were incompletely cleaved, and residues linked at both O-2 and O-6 were not cleaved at all. It was concluded that reductive cleavage is an attractive method for determination of the structure of polysaccharides.     

Linkage disequilibrium and linkage analysis of the glucose-6-phosphatase gene

Recent studies have indicated that the four most common mutations account for 78% of mutant alleles in the glucose-6-phosphatase (G6Pase) gene. A significant fraction of mutant alleles remain unidentified. Thus, informative polymorphic markers are necessary for linkage analysis in carrier testing and prenatal diagnosis in families where mutations can not be identified. The common mutations appear to be ethnic-specific, suggesting that the individual mutations may have a common founder. With the recent discovery of the nucleotide 1176 polymorphism, we have studied whether these mutations are in linkage disequilibrium with the polymorphism. The results of polymerase chain reaction/allele-specific oligonucleotide analysis show that nucleotide 1176 C is in linkage disequilibrium with mutations R83 C and R83H, and with the splicing mutation 727G→T. The 1176 T polymorphism is in linkage disequilibrium with 459insTA. A GT repeat polymorphism has also been found. However, its heterozygosity is low. The 1176 nucleotide polymorphic marker can be used in carrier and prenatal diagnosis of GSD1a families that have unidentified mutations and are informative for this marker. Received: 27 January 1998 / Accepted: 17 April 1998