Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.
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.
Koyama
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National Institute for Medical Research, Mill Hill, London, United Kingdom.
Hughes
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RC
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D001216:Asparagine; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D005353:Fibronectins / Q000378:metabolism*; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D016023:Integrins / Q000096:biosynthesis* / Q000302:isolation & purification; D007457:Iodine Radioisotopes; D007668:Kidney; D046911:Macromolecular Substances; D008361:Mannosidases / Q000037:antagonists & inhibitors; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012276:Ricin / Q000494:pharmacology
D005353:Fibronectins; D016023:Integrins; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D001216:Asparagine; D012276:Ricin; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D008361:Mannosidases