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Kim, Yun-Gon,Kim, Sun-Young,Hur, Young-Mi,Joo, Hwang-Soo,Chung, Junho,Lee, Dong-Sup,Royle, Louise,Rudd, Pauline M.,Dwek, Raymond A.,Harvey, David J.,Kim, Byung-Gee WILEY-VCH 2006 Proteomics Vol. No.
<P>The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man<SUB>3–9</SUB>GlcNAc<SUB>2</SUB>) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with α-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the α-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.</P>
Kim, Han Ie,Saldova, Radka,Park, Jun Hyoung,Lee, Young Hun,Harvey, David J.,Wormald, Mark R.,Wynne, Kieran,Elia, Giuliano,Kim, Hwa-Jung,Rudd, Pauline M.,Lee, Seung-Taek American Chemical Society 2013 JOURNAL OF PROTEOME RESEARCH Vol.12 No.8
<P>Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of <I>N</I>-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the <I>N</I>-glycan structures showed that SF9 TIMP-1 has the simplest <I>N</I>-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their <I>N</I>-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the <I>N</I>-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to <I>N</I>-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its <I>N</I>-glycans.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2013/jprobs.2013.12.issue-8/pr400276r/production/images/medium/pr-2013-00276r_0011.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr400276r'>ACS Electronic Supporting Info</A></P>
Yun-Gon Kim,Dong-Sik Shin,David J. Harvey,Hee-Jin Jeong,Kyoung-Soon Jang,Yung-Hun Yang,Chung-Gyu Park,Pauline M. Rudd,Raymond A. Dwek,Yoon-Sik Lee,Byung-Gee Kim 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Glycan recognitionleading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Firstly, total N-glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney were qualitatively and quantitatively identified by MALDI-TOF, negative ion ESI MS/MS and normal-phase HPLC (NP-HPLC) combined with exoglycosidase digestion. Over 100 N-glycans, including sialylated and neutral types, were identified. Without any derivatization steps, the characterizedpig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.