Aberrant O-Glycosylation in the Collagenous Domain of Proα2(I) Procollagen Subunits Synthesized by Chemically Transformed Hamster Fibroblasts

Chemically transformed Syrian hamster embryo fibroblasts (NQT-SHE) do not synthesize the proα1(I) subunit of type I collagen, but they secrete two forms of the proα2(I) subunit (N33 and N50) with abnormal posttranslational modifications localized in the α2CB3,5 cyanogen bromide peptide of the collagenous domain (B. Peterkofsky and W. Prather (1992)J. Biol. Chem.267 5388–5395). Isoelectric focusing and treatment of the modified chains with glycosidases and biotinylated Jacalin lectin identified the modifications as Galβ1,3GalNAc-O-Ser/Thr with or without a terminal sialic acid in an α2,6 linkage. Unhydroxylated N33 α-chains also reacted with Jacalin, confirming that the abnormal modification was O-glycosylation and not hyperhydroxylation of proline or lysine. Cells were treated with benzyl GalNAc, a competitive inhibitor of galactosyl transferase that prevents addition of Gal to GalNAc-O-Ser/Thr and thus blocks elongation of O-glycosyl chains. Treated cells secreted proα2(I) chains containing GalNAc-O-Ser/Thr but no galactose or sialic acid, which suggested that Gal addition takes place before sialylation. Treatment of NQT-SHE cells with monensin and brefeldin A inhibited secretion and led to intracellular accumulation of proα2(I) chains that contained only GalNAc. Therefore, it appears that GalNAc addition to proα2(I) chains in NQT-SHE cells occurs in thecis-Golgi, while sialic acid and galactose are added in thetrans-Golgi network. The proα2(I) chains produced by NQT-SHE cells most likely are modified because they are in the denatured state, and thus potential O-glycosylation sites become available that would not be exposed in normal triple helical procollagen.