Overglycosylation introduces local changes in triple helix alignment in collagen type I fibril structure

Collagen fibrils constitute the structural scaffold of bone, and their hierarchical organization is central to biomineralization. While hydroxylation and glycosylation of lysine residues are well-known collagen post-translational modifications, their structural consequences remain poorly understood. Here we show that excess of glycosylation of hydroxylysine residues leads to significant alterations in the local packing of collagen molecules within the fibrils. By prolonging the enzymatic modification window during helix folding through the use of cyclosporin A, an increase in double glycosylation was observed at specific sites. These overglycosylated residues were pinpointed by mass spectrometry, while cryogenic TEM revealed fibrils with reduced diameters and distinct displacements of defined sub-bands within the D-period, without altering the overall periodicity. By mapping the modified residues onto the quarter-staggered model, we have been able to correlate site-specific glycosylation with sub-band shifts, linking chemical modification to supramolecular order. These results provide molecular-level evidence that collagen glycosylation is an active determinant of fibril structure. Such insights not only advance fundamental understanding of collagen assembly but could also illuminate mechanisms underlying bone fragility disorders, including osteogenesis imperfecta, that feature altered glycosylation.