Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

Abstract

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.