The O-linked-N -acetylglucosamine (O-GlcNAc) modifi cation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the aetiology of diabetes and neurodegeneration. This intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, OGT. Here we report a novel OGT, EOGT, responsible for extracellular O-GlcNAcylation. Although both OGT and EOGT are regulated by hexosamine fl ux, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. Loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy (Dp), a membrane-anchored extracellular protein, is O-GlcNAcylated, and EOGT is required for Dp-dependent epithelial cell -matrix interactions. Thus, O-GlcNAcylation of secreted and membrane glycoproteins is a novel mediator of cell -cell or cell -matrix interactions at the cell surface.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29.7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn 2؉ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse ␤-1,4-galactosyltransferase (EC 2.4

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There was a correlation between decreased parotid gland volume and decreased saliva production in patients with head-and-neck cancer undergoing RT. Parotid volume reduction may predict parotid gland function.

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N-and C-terminal regions of the prefoldin ␤ subunit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.Molecular chaperones are ubiquitous proteins that are required for the correct folding, assembly, transport, and degradation of proteins within the cell (1). One class of chaperones, termed chaperonins, are seven-to nine-membered double ring complexes of 800 -1000 kDa that capture non-native proteins in their central cavity to promote correct folding in an ATP-dependent manner (2-5). They are classified into two groups, group I found in bacteria and organelles of eukaryotes, and group II in archaea and in the cytoplasm of eukaryotes (6, 7). The bacterial group I chaperonin, GroEL, is a cylinder-shaped tetradecamer that is capped by the heptameric co-chaperone, GroES (8 -10). In contrast, the group II chaperonin exists as an eight-or nine-rotationally symmetric double ring in a toroidal structure composed of homologous subunits of about 60 kDa and functions independently of a co-chaperone corresponding to GroES (11,12). The crystal structures of the group II chaperonins from the acidothermophilic archaeum Thermoplasma acidophilum and the hyperthermophilic archaeum Thermococcus sp. strain KS-1 suggest that the long helical protrusions located at the opening of their cavities, in their apical domains, play the equivalent role of GroES as a built-in lid of the cavity (13-15). The archaeal chaperonin takes an open conformation in the nucleotide-free or ADP-bound states and changes to a closed conformation upon binding ATP (16,17). Interestingly, closure of the built-in lid of CCT, 1 the eukaryotic cytosol group II chaperonin, is induced not by the binding but by the hydrolysis of ATP (18).The group II chaperonins cooperate with a co-chaperone, prefoldin/GimC (19 -21). Prefoldin (PFD) has been shown to participate in the maturation of actin and members of the tubulin family by transferring them in the incomplete...