Truncated N-glycans destabilize POMT1 and POMT2 but do not limit cellular O-mannosylation in HEK293 cells.

Protein O-mannosylation is initiated by the ER-resident enzymes POMT1 and POMT2, both of which carry multiple N-glycans essential for solubility and activity. Although congenital disorders of glycosylation type I (CDG-I) disrupt the early N-glycosylation process, their impact on POMT biogenesis and O-mannosylation has remained unclear. Here, we show that ALG3-dependent elongation of the N-glycan precursor is required to maintain normal abundance of POMT1 and POMT2. In ALG3-deficient HEK293 cells, POMTs carry truncated Man5-type N-glycans and their steady-state protein levels are reduced by more than half, despite unchanged transcript levels. In vitro, reduced POMT abundance resulted in a proportional decrease in mannosyltransferase activity. However, O-mannosylation in cells remained unaffected: α-dystroglycan displayed normal matriglycan extension and a highly O-mannosylated KIAA1549 reporter showed unaltered site occupancy and core structures. These data indicate that POMT activity operates with substantial catalytic reserve under basal conditions, sufficient to tolerate significant reduction in enzyme levels without impairing substrate modification. Our work identifies ALG3-mediated N-glycan maturation as a determinant of POMT stability, highlights buffering capacity that preserves substrate O-mannosylation despite reduced enzyme abundance, and provides a mechanistic framework for how N-glycosylation defects could modulate POMT-dependent glycosylation in disease.