Expression of Long-form N-Acetylglucosamine-6-O-Sulfotransferase 1 in Human High Endothelial Venules

Two members of the N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) family, GlcNAc6ST-1 and GlcNAc6ST-2, function in the biosynthesis of 6-sulfo sialyl Lewis X-capped glycoproteins expressed on high endothelial venules (HEVs) in secondary lymphoid organs. Thus, both enzymes play a critical role in L-selectin-expressing lymphocyte homing. Human GlcNAc6ST-1 is encoded by a 1593-bp open reading frame exhibiting two 5' in-frame methionine codons spaced 141 bp apart. Both resemble the consensus sequence for translation initiation. Thus, it has been hypothesized that both long and short forms of GlcNAc6ST-1 may be present, although endogenous expression of either form has not been confirmed in humans. Here, the authors developed an antibody recognizing amino acid residues between the first two human GlcNAc6ST-1 methionines. This antibody specifically recognizes the long form of the enzyme, a finding validated by Western blot analysis and immunofluorescence cytochemistry of HeLa cells misexpressing long and/or short forms of human GlcNAc6ST-1. Using this antibody, the authors carried out immunofluorescence histochemistry of human lymph node tissue sections and found endogenous expression of the long form of the enzyme in human tissue, predominantly in the trans-Golgi network of endothelial cells that form HEVs. (J Histochem Cytochem 60:397-407, 2012)ArticleJOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY. 60(5):397-407 (2012)journal articl

Physical and Functional Interactions between Uracil-DNA Glycosylase and Proliferating Cell Nuclear Antigen from the Euryarchaeon Pyrococcus furiosus*S⃞

Uracil-DNA glycosylase (UDG) is an important repair enzyme in all organisms to remove uracil bases from DNA. Recent biochemical studies have revealed that human nuclear UDG (UNG2) forms a multiprotein complex in replication foci and initiates the base excision repair pathway by interacting with proliferating cell nuclear antigen (PCNA). Here, we show the physical and functional interactions between UDG and PCNA from the hyperthermophilic euryarchaeon, Pyrococcus furiosus. The physical interaction between the two proteins was identified by a surface plasmon resonance analysis. Furthermore, the uracil glycosylase activity of P. furiosus UDG is stimulated by P. furiosus PCNA (PfuPCNA) in vitro. This stimulatory effect was observed only when wild type PfuPCNA, but not a monomeric PCNA mutant, was present in the reaction. Mutational analyses revealed that our predicted PCNA-binding region (AKTLF) in P. furiosus UDG is actually important for the interaction with PfuPCNA. This is the first report describing the functional interaction between archaeal UDG and PCNA