Collagen’s enigmatic, highly conserved <i>N</i> -glycan has an essential proteostatic function

Abstract

Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single <i>N</i>-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the <i>N</i>-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of <i>N</i>-glycosylated versus non-<i>N</i>-glycosylated collagen type-I, leaving the function of the <i>N</i>-glycan unclear. We hypothesized that the collagen <i>N</i>-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the <i>N</i>-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The <i>N</i>-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER's lectin-based chaperone machinery. Finally, we show that the C-Pro <i>N</i>-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen's previously enigmatic <i>N</i>-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.