RESUMO
[This corrects the article DOI: 10.1063/1.4922611.].
RESUMO
Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human ß-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.
Assuntos
Sialiltransferases/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Clonagem Molecular , Variação Genética/genética , Glicosilação , Células HEK293 , Humanos , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/metabolismo , Sialiltransferases/química , Sialiltransferases/genética , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
A THz-pump and x-ray-probe experiment is simulated where x-ray photoelectron diffraction (XPD) patterns record the coherent vibrational motion of carbon monoxide molecules adsorbed on a Pt(111) surface. Using molecular dynamics simulations, the excitation of frustrated wagging-type motion of the CO molecules by a few-cycle pulse of 2 THz radiation is calculated. From the atomic coordinates, the time-resolved XPD patterns of the C 1s core level photoelectrons are generated. Due to the direct structural information in these data provided by the forward scattering maximum along the carbon-oxygen direction, the sequence of these patterns represents the equivalent of a molecular movie.
RESUMO
Human ß-galactoside α-2,6-sialyltransferase I (ST6Gal-I) establishes the final glycosylation pattern of many glycoproteins by transferring a sialyl moiety to a terminal galactose. Complete sialylation of therapeutic immunoglobulins is essential for their anti-inflammatory activity and protein stability, but is difficult to achieve in vitro owing to the limited activity of ST6Gal-I towards some galactose acceptors. No structural information on ST6Gal-I that could help to improve the enzymatic properties of ST6Gal-I for biotechnological purposes is currently available. Here, the crystal structures of human ST6Gal-I in complex with the product cytidine 5'-monophosphate and in complex with cytidine and phosphate are described. These complexes allow the rationalization of the inhibitory activity of cytosine-based nucleotides. ST6Gal-I adopts a variant of the canonical glycosyltransferase A fold and differs from related sialyltransferases by several large insertions and deletions that determine its regiospecificity and substrate specificity. A large glycan from a symmetry mate localizes to the active site of ST6Gal-I in an orientation compatible with catalysis. The glycan binding mode can be generalized to any glycoprotein that is a substrate of ST6Gal-I. Comparison with a bacterial sialyltransferase in complex with a modified sialyl donor lends insight into the Michaelis complex. The results support an SN2 mechanism with inversion of configuration at the sialyl residue and suggest substrate-assisted catalysis with a charge-relay mechanism that bears a conceptual similarity to serine proteases.
Assuntos
Antígenos CD/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Sialiltransferases/química , Antígenos CD/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligação Proteica , Sialiltransferases/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Photoelectron spectroscopy (PES) is a versatile tool, which provides insight into electronic structure and dynamics in condensed matter, surfaces, interfaces and molecules. The history of PES is briefly outlined and illustrated by current developments in the field of time-resolved PES. Our group's research is mostly aimed at studying ultrafast processes and associated lifetimes related to electronic excitation at solid surfaces.