Bradley K. Hayes

O-Linked N-Acetylglucosamine: The “Yin-Yang” of Ser/Thr Phosphorylation?

O-linked N-acetylglucosamine (O-GlcNAc) was discovered during studies using bovine milk galactosyltransferase to ‘map’ the surface topography on cells of the murine immune system (Torres and Hart, 1984). Later, O-GlcNAc was shown to reside almost exclusively in the nucleus and cytoplasm (Kearse and Hart, 1991b), and to be present in eukaryotes ranging from trypanosomes, yeast, plants, to man, as well as in viruses (Hart et al. 1989; Hart et al. 1994a; Greis and Hart, 1994). We now know that O-GlcNAc is an exceedingly abundant post-translational modification of specific serine/threonine residues of many important nuclear and cytoplasmic proteins (Haltiwanger et al.1992b). Figure 1 lists the O-GlcNAc-bearing proteins identified to date. O-GlcNAc is attached as a monosaccharide and is generally not further modified. The O-GlcNAc turn-over rates are typically many-times that of the polypeptide backbone on which it is found (Chou et al.1992a; Hart and Roquemore, unpublished). The saccharide is attached at sites similar to those also used by the ‘growth-factor’, proline-directed family of kinases (Roach, 1991; Taylor and Adams, 1992). O-GlcNAc-bearing proteins have a diverse range of functions, but are characterized by several common features: 1) They all are also phosphorylated. 2) They typically form specific and regulated multimeric associations with other polypeptides. 3) In several cases, O-GlcNAcylation and phosphorylation appear to be reciprocal events.

Novel forms of protein glycosylation

Abstract Glycosylation plays several important roles in glycoprotein function. Some of these roles, such as protein folding, protein stability and efficient intracellular transport, are more dependent simply on the presence of a saccharide, rather than on its exact structure. Other functions are directly related to specific saccharide structures. This article focuses on the occurrence and potential functions of such novel and specific glycosylations as O-N -acetylglucosamine, glucose-phosphate-man nose, N -acetylgalactosamine-bearing N -linked oligosaccharides, glucosylasparagine and glycosylations of epidermal growth factor modules.

Protein O-GlcNAcylation: Potential mechanisms for the regulation of protein function

Protein O-GlcNAcylation is the process whereby single N-acetylglucosamine residues are glycosidically linked to the hydroxyl side chains of specific serine and threonine residues. O-GlcNAc was originally identified while probing the surfaces of lymphocytes using UDP-[3H] galactose and highly purified galactosyltransferase (1). O-GlcNAc was not a substrate for galactosyltransferase unless the cell membrane was first disrupted with detergents indicating that it is an intracellular glycosylation. Subcellular fractionation further demonstrated that O-GlcNAc is found exclusively on nuclear and cytosolic proteins (2,3). Galactosyltransferase labeling of mouse liver nuclei with subsequent analysis by 2-dimensional gel electrophoresis and fluorography indicates that a large number of nuclear proteins are modified with O-GlcNAc residues and suggests that O-GlcNAc is as abundant as phosphorylation (4).

Ubiquitous and temporal glycosylation of nuclear and cytoplasmic proteins

Gerzrldk& K C I I I W ~ ~ 11. Greis, I,.-Y. Dermis I h g , Melissa A. Bloinherg Teh-Yirig Chou, MatiSliiow Jiatg Llizaheth I? I ~ ~ I ~ I I I ~ ~ c , loris M. Snow, Lisa K. Kreppel, Itoberl C. Cole atid Bradley K. Hayes. Dept. Biocheni. & Molecular Genetics, University of Alabama at Birmingham, UAB Station, 1918 University Avenue, Birmingham, AL 35294-0005;Phone: (205) 934-4753; FAX: (205) 975-6685; Internet: GWHART@BMG.BHS.UAB.EDU.

HPLC methods for the fractionation and analysis of negatively charged oligosaccharides and gangliosides.

This unit describes the fractionation and analysis of anionic oligosaccharides and gangliosides using anion‐exchange high‐performance liquid chromatography (HPLC). Saccharides or gangliosides are eluted in order of the number of negative charges they possess, although the charge‐to‐mass ratio can also contribute to elution position.

Fractionation and Analysis of Neutral Oligosaccharides by HPLC

This unit describes the fractionation and analysis of neutral oligosaccharides by high‐performance liquid chromatography (HPLC) on bonded amine columns. Separation is based upon hydrogen bonding between the NH2 groups of the column and the hydroxyl groups of the oligosaccharides. A support protocol describes the reduction and desalting of neutral oligosaccharides with sodium borohydride.