Structural Analysis Of Glycans Research Articles

Human genetic deficits in glycan formation.

Glycans are associated with most proteins found in secretions and on the surface of mammalian cells. Glycans of secreted glycoproteins affect many protein properties such as solubility, stability, protease sensitivity, and polarity, while glycans on cell surface glycoproteins are involved in various cellular functions including cell-cell and cell-matrix interactions during embryogenesis, immune reactions, and tumor development. Recent advances in human genomic research together with newly developed and sensitive methods for the analysis of glycan structures have elucidated the etiology of a growing number of human genetic diseases with aberrant glycan formation. Among these diseases, defects of protein N-glycosylation and O-mannosylation are reviewed here. The former is relatively common and the latter is rather uncommon. Both types of defects lead to severe abnormalities, which indicate the importance of glycosylation. Sequencing of the human genome is essentially complete and now glycobiology becomes an important area of postgenomic research. Glycobiology is expected to produce remarkable advances in the understanding and treatment of certain genetic diseases. (Communicated by Tamio YAMAKAWA, M.J.A.)

Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to the 120 kDa APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analysed the various glycans on the 120 kDa APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of the 4 possible N-glycosylation sites are occupied with fucosylated paucimannose {Man 2–3(Fuc 1–2)GlcNAc 2-peptide} type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal “stalk” region of the protein. It is likely that 5–6 of the 13 sites are occupied, probably with simple {GalNAc-peptide} type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc rich, is the most likely binding site for Cry1Ac.

Introduction to glycobiology

1. Concepts of Glycobiology 2. N-linked glycosylation 3. Conformations of oligosaccharides 4. Strategies for analysis of glycan structures 5. O-linked glycosylation 6. Glycolipids and membrane glycosylation 7. Effects of glycosylation on protein structure and function 8. Glycoprotein trafficking in cells and organisms 9. Carbohydrate recognition in cell adhesion and signalling 10. Mechanisms of sugar recognition in animal lectins 11. Glycobiology of plants, bacteria and viruses 12. Glycosylation and disease 13. The future of glycobiology

Glycosylation in the control of selectin counter-receptor structure and function.

Leukocyte trafficking is characterized by sequential cell adhesion and activation events that deliver specific leukocyte subsets to distinct extravascular locations under different pathophysiological circumstances. E-, P- and/or L-selectin-dependent leukocyte-endothelial cell adhesive interactions contribute essentially to this process. Selectin counter-receptor activity on leukocyte and high endothelial venules is borne by specific glycoproteins whose ability to support adhesion requires specific post-translational modifications. These modifications are typified by serine/threonine-linked oligosaccharides capped with the sialyl Lewis x moiety, an alpha2-3sialylated, alpha1-3ucosylated tetrasaccharide synthesized by specific glycosyltransferases. Recent advances in glycan structure analysis and in characterizing mice with targeted deletions of glycosyltransferase and sulfotransferase genes discloses an essential role for 6-O GlcNAc sulfate modification of the sialyl Lewis x tetrasaccharide in L-selectin counter-receptor activity. Related studies identify novel extended Core 1 type O-glycans bearing the 6-sulfosialyl Lewis x moiety, define the molecular nature of the MECA-79 epitope, and disclose a requirement for the alpha1-3fucosyltransferases FucT-IV and FucT-VII in the elaboration of L-selectin counter-receptor activities. Parallel studies also demonstrate that these 2 fucosyltransferases, a core 2 GlcNAc transferase, and core 2-type sialyl Lewis x determinants make essential contributions to leukocyte P-selectin counter-receptor activity, and figure prominently in the control of leukocyte E-selectin counter-receptor activity.

Congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are a rapidly growing group of genetic diseases that are due to defects in the synthesis of glycans and in the attachment of glycans to other compounds. Most CDG are multisystem diseases that include severe brain involvement. The CDG causing sialic acid deficiency of N-glycans can be diagnosed by isoelectrofocusing of serum sialotransferrins. An efficient treatment, namely oral D-mannose, is available for only one CDG (CDG-Ib). In many patients with CDG, the basic defect is unknown (CDG-x). Glycan structural analysis, yeast genetics, and knockout animal models are essential tools in the elucidation of novel CDG. Eleven primary genetic glycosylation diseases have been discovered and their basic defects identified: six in the N-glycan assembly, three in the N-glycan processing, and two in the O-glycan (glycosaminoglycan) assembly. This review summarizes their clinical, biochemical, and genetic characteristics and speculates on further developments in this field.