Current Drug Targets - Volume 9, Issue 4, 2008

Glycoconjugates (glycolipids, glycoproteins and proteoglycans) are ubiquitous on eukaryotic cell surfaces. Isolation, chemical structure determinations, and characterization of individual Glycosyl-transferases for their biosynthesis took almost 4 decades (until the early 1990's) of hard work by endless glycobiochemists and glycobiologists around the world. From 1986 until now putative gene sequences of almost 300 glycosyltransferases have been established. Each glycoconjugate is characterized by its unique structure synthesized by activities of a group of specific glycosyltransferases. It is nearly established that a specific linkage (between two sugars) is established by the catalysis of a specific gene product (or a glycosyltransferase) as proposed by Professor Saul Roseman in early 1970's; sometimes with little variation of gene sequence, a different linkage formation is also catalyzed as observed recently. However, many questions remain unanswered: i) what are the active sites of these enzymes present in the protein sequences? ii) How are these enzymes transcriptionally regulated? iii) How are these glycosyltransferases post-translationally modified and regulated? iv) How are these glycosyltransferases regulated in apoptotic and metastatic cells? When our chief editor contacted me two years ago to edit an issue on “Glycosyltransferases” in this new well-rated journal, I agreed to do so, keeping these questions in my mind. At first I had to identify all those excellent scientists who are actively contributing in this field of “Glycosyltransferases” and secondly to propose to each individual author (also with their active collaborators) to write an article wherein, with their expertise, they could write for the interested readers the latest words in which that area in which they are working. Finally, after one year of negotiations I received 13 well-written articles from the experts in the field, which covered some of the questions raised earlier. We decided to publish these thirteen articles in two volumes. Of course many active researchers in this field were unable to contribute in these two volumes. To maintain the high standard of this journal I went through every line of each article and checked almost every reference so that it is correctly quoted in the right place; of course this was a time consuming task for one person. Finally, I am able to release the first volume of the “Glycosyltransferases” in this journal, with six papers to the press, and we are almost ready within a few months to send to press the all other 7 articles to be printed in the second volume. A total list of all the accepted all 13 articles will be printed in both the volumes. I am happy to accept the responsibility as a special editor for these two volumes. The chief editor is my good friend for 37 years who requested me to edit and I am also expecting to have a few more new friends for rest of my life after these two volumes are published.

Mannosylphospho dolichol synthase (DPMS) catalyzes the transfer reaction GDP-mannose + Dol-P ?? Dol-PMan + GDP, a ‘key step’ in the assembly of lipid-linked oligosaccharide (LLO) and a pre-requisite for asparagine-linked (N-linked) protein glycosylation. DPMS is present from a protozoan parasite to human, and its sequence carries a cAMPdependent phosphorylation motif. We have evaluated the involvement of DPMS in angiogenesis, an essential physiological event during the growth of breast and other solid tumors. It has been observed that enhancers of intracellular cAMP accelerated the capillary endothelial cell proliferation by reducing the cell cycle duration. Reduced Con A to WGA fluorescence ratio indicated high level complex type N-glycans on the cell surface. This was supported by upregulated LLO biosynthesis in cells stimulated either with a β-agonist isoproterenol or other cAMP enhancer, such as 8Br-cAMP, forskolin, cholera toxin, or prostaglandin E1. The turnover (t1/2) of LLO was also increased. Increased LLO biosynthesis correlated extremely well with the DPMS activity in cells treated with 8Br-cAMP. High DPMS activity in isoproterenoltreated cells was not due to an increased gene expression because actinomycin D failed to block the upregulation. cDNA cloning of capillary endothelial cell Dpm1 gene and the deduced amino acid sequence identified a PKA motif in capillary endothelial cell DPMS. Thus, it has been concluded that increased DPMS activity through protein phosphorylation is a driving force for angiogeneis. Its abolition, however, led to cell arrest in G1 and induction of apoptosis.

Lactosylceramide is a ubiquitously present glycosphingolipid in mammalian tissues and has been implicated in cell proliferation, adhesion, migration and angiogenesis. This glycosphingolipid is synthesized by Golgi-localized enzyme LacCer synthase. According to recent nomenclature and gene mapping studies, two LacCer synthases β1,4GalT-V and β1,4GalT-VI have been identified and characterized. In addition, β1,4GalT-V has been implicated in the synthesis of Nglycans of cell surface glycoproteins. During the past two decades data have accumulated suggesting that the cellular level of LacCer can be regulated by various growth factors, cytokines, lipids, lipoproteins and hemodynamic factors, such as fluid shear stress, by altering the activity of LacCer synthase. An interesting feature is that a nuclear regulating factor (SP1) plays a critical role in transcriptional regulation of this enzyme in cancer cells. Moreover, in human umbilical vein endothelial cells, NF-κB has been also shown to regulate this enzyme which, in turn, regulates the gene/protein expression of platelet endothelial cell adhesion molecule, intercellular cell adhesion molecule and angiogenesis. Since new blood supply via formation of capillaries is critical in tumor growth, metastasis, and atherogenesis, these findings expand the role of enzyme in these pathologies. Additional studies are warranted to understand the molecular and biochemical basis of how LacCer synthases are regulated. These studies will facilitate advances in discovery of drugs which mitigate diseases, such as atherosclerosis and cancer due to an aberrant regulation of these LacCer synthases.

Gangliosides make up a group of sialic acid-containing complex glycosphingolipids particularly abundant in the central nervous system. The finding indicating gangliosides are stored in certain hereditary diseases affecting the central nervous system opened the interest in studying their metabolism. The initial in vitro pioneering work on the glycosyltransferases involved in ganglioside biosynthesis was done by Roseman and his associates primarily in embryonic chick brains almost forty years ago. Since that time enzymes catalyzing the formation of main human gangliosides have been successfully purified and cloned. Their specificity has been determined and their subcellular localization and topology has been established. Transgenic mouse models deficient in distinct ganglioside-directed glycosyltransferases are available and represent a vital step toward understanding the metabolism and function of this challenging lipid class. In the present review we briefly introduce the reader in the complex structure of gangliosides, then we summarize new developments concerning their function especially regarding neurodegenerative disorders, and in this article we would like to review on what is known about glycosyltransferases that catalyze the formation of these complex lipids in the Golgi apparatus, that was established by Basu and his associates almost three decades ago.

Beta-1,4-galactosylransferase (β4Gal-T1) participates in the synthesis of Galβ1-4-GlcNAc-disaccharide unit of glycoconjugates. It is a trans-Golgi glycosyltransferase (Glyco-T) with a type II membrane protein topology, a short Nterminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain facing the trans-Golgi-lumen. Its hydrophobic membrane-spanning region, like that of other Glyco-T, has a shorter length compared to plasma membrane proteins, an important feature for its retention in the trans-Golgi. The catalytic domain has two flexible loops, a long and a small one. The primary metal binding site is located at the N-terminal hinge region of the long flexible loop. Upon binding of metal ion and sugar-nucleotide, the flexible loops undergo a marked conformational change, from an open to a closed conformation. Conformational change simultaneously creates at the C-terminal region of the flexible loop an oligosaccharide acceptor binding site that did not exist before. The loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. The conformational change in β4Gal-T1 also creates the binding site for a mammary gland-specific protein, α-lactalbumin (LA), which changes the acceptor specificity of the enzyme toward glucose to synthesize lactose during lactation. The specificity of the sugar donor is generally determined by a few residues in the sugar-nucleotide binding pocket of Glyco-T, conserved among the family members from different species. Mutation of these residues has allowed us to design new and novel glycosyltransferases, with broader or requisite donor and acceptor specificities, and to synthesize specific complex carbohydrates as well as specific inhibitors for these enzymes.

Six human β-galactoside α2,3-sialyltransferase genes, which are hST3Gal I-VI, have been cloned. Multiple genes encode enzymes with closely related catalytic specificities but different patterns of tissue expression. The multiple genes correspond to the control of various tissue specific regulators. Several studies have examined the transcriptional regulation of some human β-galactoside α2,3-sialyltransferases genes. Multiple mRNA forms differing only in the 5'- untranslated regions have been identified in hST3Gal II, hST3Gal III, hST3Gal IV, hST3Gal V, and hST3Gal VI. These transcripts are produced by a combination of alternative splicing and promoter utilization, suggesting the transcriptional regulation of this gene depends on the use of alternative promoters, further suggesting that tissue-specific transcriptional regulation of these genes depends on the use of multiple genes and multiple promoters. The multiple regulatory pathways of these ubiquitous sialyltransferases may be differentially modulated in various cell types.

Ganglioside synthases are glycosyltransferases involved in the biosynthesis of glycoconjugates. A number of ganglioside synthase genes have been cloned and characterized. They are classified into different families of glycosyltransferases based on similarities of their amino acid sequences. Tissue-specific expression of these genes has been analyzed by hybridization using cDNA fragments. Enzymatic characterization with the expressed recombinant enzymes showed these enzymes differ in their donor and acceptor substrate specificities and other biochemical parameters. In vitro enzymatic analysis also showed that one linkage can be synthesized by multiple enzymes and one enzyme may be responsible for synthesis of multiple gangliosides. Following the cloning of the ganglioside synthase genes, the promoters of the key synthase genes in the ganglioside biosynthetic pathway have been cloned and analyzed. All of the promoters are TATA-less, lacking a CCAAT box but containing GC-rich boxes, characteristic of the house-keeping genes, although transcription of ganglioside synthase genes is subject to complex developmental and tissue-specific regulation. A set of cis-acting elements and transcription factors, including Sp1, AP2, and CREB, function in the proximal promoters. Negative- regulatory regions have also been defined in most of the promoters. We present here an overview of these genes and their transcriptional regulation.

Aging has become a major health care problem and socio-economic burden worldwide. Myocardial infarction (MI) is the major killer worldwide and coronary reperfusion is the major form of acute post-MI therapy. The aging population is increasing, and with it, morbidity and mortality due to impaired healing after ST-segment elevation MI (STEMI) and its consequences. Optimal healing of the wounded heart is critical for preservation of structural and functional integrity of the pumping chambers, survival, and a favorable outcome irrespective of age. Although STEMI is more prevalent in the elderly and impaired healing during aging may promote adverse remodeling and thereby jeopardize outcome, there is an information gap on post-STEMI healing and its therapy in the elderly. Current therapies during post-STEMI healing are aimed primarily at the <65 age-group and preclinical studies tend to test drugs in mostly young animals. Therapies over the last decade have improved post-MI survival mainly in patients aged < 65 years. Novel healing-specific proteins may provide potential targets for improving healing and limiting adverse remodeling of the post-STEMI heart in the elderly, thereby improving outcome.

The active metabolite of vitamin D3 - 1,25-(OH)2D3 - exerts most of its physiological and pharmacological actions through its nuclear receptor (VDR), regulating the transcriptional machinery of a variety of cell types. Basic research motivated by the detection of VDR in numerous target cells, has indicated potential therapeutic applications of VDR ligands in osteoporosis, cancer, secondary hyperparathyroidism and autoimmune diseases such as psoriasis, systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes and multiple sclerosis. In recent years vitamin D analogs, particularly calcipotriol and tacalcitol, have been used as topical therapeutic agents in vitiligo, an autoimmune pigmentary disorder characterized by aberrant loss of functional melanocytes from involved epidermis. The presence of cytotoxic T cells targeting melanocyte antigens and imbalance of the cytokine network were described as characteristics of the disease, eventually leading to melanocyte damage and death. Vitamin D ligands are designed to target the local immune response in vitiligo, acting on specific T cell activation, mainly by inhibiting the transition of T cells from early to late G1 phase and by inhibiting the expression of several pro-inflammatory cytokines genes, such as those encoding tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). Vitamin D3 compounds are known to influence melanocyte maturation and differentiation and also to up-regulate melanogenesis through pathways activated by specific ligand receptors, such as endothelin receptor and c-kit. In this review we summarize the complex pathogenetic rationale of vitamin D analogs in vitiligo depigmentation. Understanding the cellular and molecular mechanisms through which vitamin D targets the epidermal melanin unit is of great interest for identification of new effective therapeutic combination(s) that might induce repigmentation in vitiligo.