No abstract

Background: Kunitz-type inhibitors provide a suitable scaffold for novel elastase inhibitors. Results: The inhibitor ShPI-1 was modified for pancreatic elastase binding, and the crystal structure of the complex was elucidated and analyzed. Conclusion: The extended protease-inhibitor interactions provide a potential switch to direct inhibitor selectivity toward elastases. Significance: These results will help to design novel elastase inhibitors for the treatment of tissue destruction diseases.

Terminally sialylated oligosaccharides were synthesised employing recombinant trans-sialidase from Trypanosoma cruzi. Regio- and stereoselectively Sia-alpha(2-3)-Gal-betaR derivatives could be obtained in respectable yields, using combined chemical and enzymatic methodologies. An array of different disaccharide precursors such as Gal-beta(1-3)-GalNAc-alphaSer/Thr, lactosides and lactosamide derivatives were sialylated and successfully purified by facile isolation procedures. Depending on the acceptor structure isolated, yields for trans-sialylation products were between 20 and 60%.

Recombinant transsialidase from Trypanosoma cruzi (TcTS) was used for the sialylation with natural and non-natural derivatives of neuraminic acid. Neu5Ac-alpha(2-->3)-Gal-beta(1-->6)-Glc-alphaOMe was prepared in 80 % yield. Correspondingly, the modified trisaccharide derivatives Neu5Prop-alpha(2-->3)-Gal-beta(1-->6)-Glc-alphaOMe (32 %) and Neu5Gc-alpha(2-->3)-Gal-beta(1-->6)-Glc-alphaOMe (Prop=propanoyl, Gc=glycolyl) were obtained in 60 % yield, respectively.

Human α1,3-fucosyltransferase IX catalyzes the transfer of l-fucose from guanosine diphosphate-β-L-fucose to N-acetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential N-glycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed ∼60% of wt activity and N101Q retained ∼30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.