A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae.

We describe a novel synthetic N-glycosylation pathway to produce recombinant proteins carrying human-like N-glycans in Saccharomyces cerevisiae, at the same time addressing glycoform and glycosylation efficiency. The Δalg3 Δalg11 double mutant strain, in which the N-glycans are not matured to their native high-mannose structure, was used. In this mutant strain, lipid-linked Man(3)GlcNAc(2) is built up on the cytoplasmic side of the endoplasmic reticulum, flipped by an artificial flippase into the ER lumen, and then transferred with high efficiency to the nascent polypeptide by a protozoan oligosaccharyltransferase. Protein-bound Man(3)GlcNAc(2) serves directly as a substrate for Golgi apparatus-targeted human N-acetylglucosaminyltransferases I and II. Our results confirmed the presence of the complex human-like N-glycan structure GlcNAc(2)Man(3)GlcNAc(2) on the secreted monoclonal antibody HyHEL-10. However, due to the interference of Golgi apparatus-localized mannosyltransferases, heterogeneity of N-linked glycans was observed.

L-selectin--a dynamic regulator of leukocyte migration.

The leukocytic cell adhesion receptor L-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance. Distinct domains of L-selectin contribute to proper leukocyte migration. In this review, we discuss the contributions of these domains with respect to L-selectin function: the regulation by serine phosphorylation of the cytoplasmic tail, the role of the transmembrane domain in receptor positioning on the cell surface as well as the N-glycosylation of the extracellular part and the identification of novel binding partners.

Inhibition of L-selectin binding by polyacrylamide-based conjugates under defined flow conditions.

Selectins mediate tethering and rolling of leukocytes along the endothelium in a shear force-dependent manner. This key step in the cellular immune response is a target for experimental anti-inflammatory therapies. In the present paper we have examined the inhibitory activity of the minimal selectin ligand sialyl Lewis x (SiaLe(x)), its isomer sialyl Lewis a (SiaLe(a)) and sulfated tyrosine (sTyr) residues under dynamic flow reflecting the rheological conditions in the blood stream. The monomeric ligands were compared to multivalent polyacrylamide (PAA)-based conjugates under defined flow conditions on the molecular level, using surface plasmon resonance (SPR) technology, and on the cellular level, using a parallel-plate flow chamber. SPR measurements showed that a spatial arrangement of binding epitopes mimicking the selectin binding motif of the natural ligand PSGL-1 inhibits L-selectin binding successfully with IC(50) values in the nanomolar range. Using a flow chamber adhesion assay it could be shown that the multivalent inhibitors efficiently blocked rolling and tethering of NALM-6 pre-B cells transfected with human L-selectin to activated endothelium and that the inhibitory activity increased with rising shear stress. While PAA-conjugates were almost not inhibitory at low shear stress, NALM-6 cell rolling was nearly completely inhibited at high shear stress. The results indicate that multimeric conjugates of SiaLe(x), SiaLe(a) and sTyr are highly effective inhibitors of L-selectin-mediated cell adhesion particularly under flow conditions. Consequently, SiaLe(x), SiaLe(a) and/or sTyr on macromolecular carriers may be promising candidates for anti-inflammatory therapy.