Donor substrate binding to trans-sialidase of Trypanosoma cruzi as studied by STD NMR

2007 ◽  
Vol 342 (12-13) ◽  
pp. 1904-1909 ◽  
Author(s):  
Astrid Blume ◽  
Björn Neubacher ◽  
Joachim Thiem ◽  
Thomas Peters
2011 ◽  
Vol 9 (5) ◽  
pp. 1653 ◽  
Author(s):  
Jennifer A. Harrison ◽  
K. P. Ravindranathan Kartha ◽  
Eric J. L. Fournier ◽  
Todd L. Lowary ◽  
Carles Malet ◽  
...  

Author(s):  
Michael A. Järvå ◽  
Marija Dramicanin ◽  
James P. Lingford ◽  
Runyu Mao ◽  
Alan John ◽  
...  

AbstractFucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique conformational change associated with donor substrate binding; common strategies employed by fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.


2007 ◽  
Vol 177 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Daniel J. Kelleher ◽  
Sulagna Banerjee ◽  
Anthony J. Cura ◽  
John Samuelson ◽  
Reid Gilmore

The dolichol-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol is the in vivo donor substrate synthesized by most eukaryotes for asparagine-linked glycosylation. However, many protist organisms assemble dolichol-linked oligosaccharides that lack glucose residues. We have compared donor substrate utilization by the oligosaccharyltransferase (OST) from Trypanosoma cruzi, Entamoeba histolytica, Trichomonas vaginalis, Cryptococcus neoformans, and Saccharomyces cerevisiae using structurally homogeneous dolichol-linked oligosaccharides as well as a heterogeneous dolichol-linked oligosaccharide library. Our results demonstrate that the OST from diverse organisms utilizes the in vivo oligo saccharide donor in preference to certain larger and/or smaller oligosaccharide donors. Steady-state enzyme kinetic experiments reveal that the binding affinity of the tripeptide acceptor for the protist OST complex is influenced by the structure of the oligosaccharide donor. This rudimentary donor substrate selection mechanism has been refined in fungi and vertebrate organisms by the addition of a second, regulatory dolichol-linked oligosaccharide binding site, the presence of which correlates with acquisition of the SWP1/ribophorin II subunit of the OST complex.


2020 ◽  
Vol 295 (19) ◽  
pp. 6677-6688 ◽  
Author(s):  
Michael A. Järvå ◽  
Marija Dramicanin ◽  
James P. Lingford ◽  
Runyu Mao ◽  
Alan John ◽  
...  

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.


2012 ◽  
Vol 1820 (12) ◽  
pp. 1915-1925 ◽  
Author(s):  
Miriam P. Kötzler ◽  
Simon Blank ◽  
Frank I. Bantleon ◽  
Edzard Spillner ◽  
Bernd Meyer

Glycobiology ◽  
2000 ◽  
Vol 10 (5) ◽  
pp. 503-510 ◽  
Author(s):  
T. Takahashi ◽  
Y. Ikeda ◽  
A. Tateishi ◽  
Y. Yamaguchi ◽  
M. Ishikawa ◽  
...  

Author(s):  
Irwin I. Singer

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.


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