ABERRANT O-GLYCOSYLATION IN THE COLLAGENOUS DOMAIN OF PRO-ALPHA-2(I) PROCOLLAGEN SUBUNITS SYNTHESIZED BY CHEMICALLY TRANSFORMED HAMSTER FIBROBLASTS

Chemically transformed Syrian hamster embryo fibroblasts (NQT-SHE) do not synthesize the pro alpha 1(I) subunit of type I collagen, but they secrete two forms of the pro alpha 2(I) subunit (N33 and N50) with ab normal posttranslational modifications localized in the alpha 2CB3,5 c yanogen 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 biotinylate d Jacalin lectin identified the modifications as Gal beta 1,3-GalNAc-O -Ser/Thr with or without a terminal sialic acid in an alpha 2,6 linkag e. Unhydroxylated N33 alpha-chains also reacted with Jacalin, comfirmi ng that the abnormal modification was O-glycosylation and not hyperhyd roxylation of proline or lysine, Cells were treated with benzyl GalNAc , a competitive inhibitor of galactosyl transferase that prevents addi tion of Gal tee GalNAc-O-Ser/Thr and thus blocks elongation of O-glyco syl chains, Treated cells secreted pro alpha 2(l) chains containing Ga lNAc-O-Ser/Thr but no galactose or sialic acid, which suggested that G al addition takes place before sialylation. Treatment of NQT-SHE cells with monensin and brefeldin A inhibited secretion and led to intracel lular accumulation of pro alpha 2(I) chains that contained only GalNAc . Therefore, it appears that GalNAc addition to pro alpha 2(I) chains in NQT-SHE cells occurs in the cis-Gogli, while sialic acid and galact ose are added in the trans-Golgi network. The pro alpha 2(I) chains pr oduced by NQT-SHE cells most likely are modified because they are in t he denatured state, and thus potential O-glycosylation sites become av ailable that would not be exposed in normal triple helical procollagen . (C) 1997 Academic Press.