αIIbβ3 biogenesis is controlled by engagement of αIIb in the calnexin cycle via the N15-linked glycan

Although much is known about alpha IIlb beta 3 structure and function, relatively little is understood about its biogenesis. Thus, we studied the kinetics of pro-alpha IIb production and degradation, focusing on whether proteasomal degradation or the calnexin cycle participates in these processes. In pulse-chase analyses, the time to half-disappearance of pro-alpha IIb (t1/2) was the same in (1) HEK293 cells transfected with (a) alpha IIb plus beta 3, (b) alpha IIb alone, (c) mutant V298F alpha IIb plus beta 3, or (d) I374T alpha IIb plus 03; and (2) murine wild-type and beta 3-null megakaryocytes. Inhibition of the proteasome prolonged the t1/2 values in both HEK293 cells and murine megakaryocytes. Calnexin coprecipitated with alpha IIb from HEK293 cells transfected with alpha IIb alone, alpha IIb plus beta 3, and V298F alpha IIb plus beta 3. For proteins in the calnexin cycle, removal of the terminal mannose residue of the middle branch of the core Winked glycan results in degradation. Inhibition of the enzyme that removes this mannose residue prevented pro-alpha IIb degradation in beta 3-null murine megakaryocytes. alpha IIb contains a conserved glycosylation consensus sequence at N15, and an N15Q mutation prevented pro-alpha IIb maturation, complex formation, and degradation. Our findings suggest that pro-alpha IIb engages the calnexin cycle via the N15 glycan and that failure of pro-alpha IIb to complex normally with beta 3 results in proteasomal degradation.