β-Xylosides and other glycosides prime the synthesis of protein-free glycan chains when fed to cells. To co-localize enzymes that initiate and extend these chains, we incubated these freely permeable acceptors with intact functional Golgi that transport exogenously supplied UDP-[3H]Gal. Golgi compartments having the appropriate transferases and sugar nucleotide transporters glycosylate the added acceptors, and since these Golgi do not carry out intervesicular transport, any additional glycosylations of the added acceptor must occur in same sealed compartment. This gives evidence for co-localization of additional transferases in the same compartment. In addition, some of the transferases synthesizing other glycoconjugates are located in the same Golgi subcompartments and must compete for transported sugar donors. Labeling of added acceptors consumes the limited amount of donors and leaves less available for glycosylating endogenous acceptors in the same compartment. Careful structural analysis of the glycans made in the presence and absence of added acceptors shows which transferases are co-localized. This review highlights successes and problems encountered using these two approaches. Together they offer an opportunity to do high resolution mapping of the functional Golgi within the cis, medial and trans continental borders.
Preparations and applications of a rapidly expanding family of neoglycoconjugates, recently defined as “glycopolymers”, are described. A survey of the most commonly used synthetic strategies will be discussed, together with their advantages and limitations. Incorporation of simple and complex oligosaccharide sequences into glycopolymers will be illustrated. Additionally, some chemo-enzymatic glycosylations of pre-made glycopolymers will be presented with examples that include trans-sialidase and endo-β-D-N-acetylglucosaminidase. Fundamental physical and biochemical properties of glycopolymers will be evaluated considering their designated applications. Many advantages of glycopolymers over other neoglycoconjugates have surfaced in recent years and a brief summary will be highlighted. As the applications of glycopolymers are rapidly flourishing, their syntheses in forms suitable to specific needs have been foreseen. Consequently, glycopolymers have been synthesized with tailor-made properties by strategies that involve glycopolymers syntheses with probes or effector molecules built-in. Finally, glycopolymers with specific carbohydrate residues will be used to better describe the wide variety of applications in which they are implicated. Glycopolymers used in cell cultures, epitope characterization, antibody isotyping, immuno-diagnostics, cell targeting, and in inhibition of adherence of pathogens such as influenza viruses will be described. Some recent applications suggest that glycopolymers can also be used for the prognosis of breast cancer proliferation and, in some cases, as anti-inflammatory agents.
The interest in insect glycosylation has been heightened by the biotechnological prospects of the insect cell/baculovirus system for the production of recombinant glycoproteins. It is evident that insects produce, in addition to the ubiquitous oligomannosidic N-glycans, unique structures not found in mammalian or plant glycoproteins. However, there are structural similarities to plant glycoproteins, such as α1, 3-fucosylation of the asparagine-bound GlcNAc residue, leading to immunological cross-reactions between plant and insect glycoproteins. The first steps of N-glycan biosynthesis appear to be highly conserved throughout eukaryotic cells, insects not being an exception. Thus, transfer of Glc3Man9GlcNAc2 from dolichol to protein in the endoplasmic reticulum is followed by deglucosylation and transient re-glucosylation by a glucosyltransferase acting only towards incorrectly folded glycoproteins. Mannosidase trimming then leads to oligomannosidic structures and eventually permits the action of GlcNAc-transferase I. This step turns the oligosaccharide into a substrate for α-mannosidase II. Depending on the cell line or tissue, α1, 3-and/or α1, 6-fucosyltransferases or GlcNAc-transferase II may now enter the scene. At least in honeybees there are additional transferases generating a GalNAcβ1 →4(Fucα1→3)GlcNAc antenna. In a locust, the non-sugar substituent 2-aminoethylphosphonate was found. The action of sialyltransferases or galactosyl-transferase acting on N-glycans has hitherto not been confirmed. There is now good evidence that in most insects and cell lines, the GlcNAc provided by GlcNAc-transferase I is finally removed by a membrane-bound and branch specific β-N-acetylglucosaminidase.
N-Linked glycosylation is one of the most common protein modifications and can have profound effects on protein expression and function. These effects often depend on the number and position of N-linked oligosaccharides added to a protein during core-glycosylation. While the sequon, Asn-X-Ser/Thr, serves as a recognition signal for core-glycosylation, other protein signals must also control this process, since many Asn-X-Ser/Thr sequons are glycosylated inefficiently or are not glycosylated at all. A variety of experimental approaches have been used to define the signals which control core-glycosylation at specific Asn residues. These include comparisons of protein sequences near glycosylated and non-glycosylated sequons, use of sequon-containing peptides as oligosaccharide acceptors or inhibitors, and analysis of the glycosylation of sequons in recombinant proteins. Such studies reveal that a complex set of factors determines the efficiency of oligosaccharide addition at each Asn-X-Ser/Thr sequon. Amino acids near the Asn residue in a sequon can profoundly affect its oligosaccharide acceptor activity. A variety of other factors further modulate core-glycosylation efficiency by influencing the accessibility of sequons for glycosylation at a critical time during protein synthesis. Studies addressing these issues are reviewed.