Aberrant O-glycosylation in the collagenous domain of pro alpha2(I) procollagen subunits synthesized by chemically transformed hamster fibroblasts

Chemically transformed Syrian hamster embryo fibroblasts (NQT-SHE) do not synthesize the pro alpha1(I) subunit of type I collagen, but they secrete two forms of the pro alpha2(I) subunit (N33 and N50) with abnormal post-translational modifications localized in the alpha2CB3,5 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 Gal beta1,3-GalNAc-O-Ser/Thr with or without a terminal sialic acid in an alpha2,6 linkage. Unhydroxylated N33 alpha-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 pro alpha2(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 pro alpha2(I) chains that contained only GalNAc. Therefore, it appears that GalNAc addition to pro alpha2(I) chains in NQT-SHE cells occurs in the cis-Golgi, while sialic acid and galactose are added in the trans-Golgi network. The pro alpha2(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.