Characterization of a post-translational modification of the PRO-alpha-2(I) subunit of type I procollagen synthesized by chemically transformed SHE-cells

NQT-SHE cells derived from Syrian hamster embryo (SHE) fibroblasts transformed by 4-nitroquinoline-1-oxide synthesize two modified $\alpha$2(I) subunits of type I collagen designated as N-33 and N-50 (N-collagens). The N-collagens migrate more slowly than the normal $\alpha$2(I) chain during sodium dodecyl-sulfate polyacrylamide electrophoresis (SDS-PAGE) due to an abnormal post-translational modification and not to over-hydroxylation of proline and lysine residues. The modification was not characterized but it was localized in a cyanogen bromide peptide (CB3,5) derived from the C-terminal half of the $\alpha$2(I) chain and in two peptides (V8-1 and V8-2) obtained by V8 protease digestion of the modified $\alpha$2(I) chain or the CB3,5 peptide (Peterkofsky, B. and Prather, W. (1986), J. Biol. Chem., 261, 16818-16826; (1992), J. Biol. Chem., 267, 5388-5395). The present experiments showed by isoelectric focusing that the modification was acidic and treatment of the N33$\alpha$ and N50$\alpha$ chains with linkage-specific neuraminidase showed that acidicity was due to the presence of sialic acid in an $\alpha 2\to 6$ linkage to Gal or GalNac. Treatment of the N-collagens with glycosidases identified the modification as serine or threonine linked O-glycosyl groups containing sialic acid. Removal of the O-glycosyl groups from the N-collagens caused them to migrate similarly to the normal $\alpha$2(I) chain during SDS-PAGE. The $\alpha$ chains and purified CB3,5 peptides from the N-collagens reacted with biotinylated Jacalin lectin providing additional evidence for O-glycosylation. Secretion of a pro$\alpha$2(I) monomer is unexpected since the $\lbrack\alpha 2(I)\rbrack\sb3$ homotrimer is unstable and the monomer is usually degraded intracellularly. To investigate the possibility that abnormal N-collagens are secreted and not degraded because they contain O-glycosyl groups, cells were treated with benzylGalNac which inhibits O-glycosyl chain elongation. BenzylGalNac treatment of NQT-SHE cells led to the accumulation of N33$\alpha$ and N50$\alpha$ chains containing O-GalNac but neither synthesis nor secretion was affected. The subcellular site of modification was identified by addition of monensin and brefeldin A, which block the secretory pathway. By the use of neuraminidase and O-glycanase enzyme treatment and Jacalin binding it was found that the N-collagens that accumulated intracellularly in the presence of the inhibitors contained GalNac but neither galactose nor sialic acid. These results suggest that addition of GalNac to the $\alpha$2(I) chain occurs in the cis-Golgi while galactose and sialic acid may be added in the trans-Golgi network.