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Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both N- and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts 3 weeks later. In contrast, inhibition of N-glycan processing in MSCs altered differentiation to enhance the mineralization capacity of the osteoblasts. The effect of N-glycans on MSC differentiation was mediated by the phosphoinositide-3-kinase (PI3K)/Akt pathway owing to reduced Akt phosphorylation. Interestingly, by inhibiting PI3K during the first 2 days of osteogenesis, we were able to phenocopy the effect of inhibiting N-glycan processing. Thus, glycan processing provides another layer of regulation that can modulate the functional outcome of differentiation. Glycan processing can thereby offer a novel set of targets for many therapeutically attractive processes.

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.