Characterization of a post-translational modification of the PRO-alpha-2(I) subunit of type I procol...

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" Characterization of a post-translational modification of the PRO-alpha-2(I) subunit of type I procollagen synthesized by chemically transformed SHE-cells "
U. Ramachandran A. Cheh

Document Type	:	Latin Dissertation
Language of Document	:	English
Record Number	:	1113352
Doc. No	:	TLpq304171826
Main Entry	:	A. Cheh
	:	U. Ramachandran
Title & Author	:	Characterization of a post-translational modification of the PRO-alpha-2(I) subunit of type I procollagen synthesized by chemically transformed SHE-cells\ U. RamachandranA. Cheh
College	:	American University
Date	:	1995
student score	:	1995
Degree	:	Ph.D.
Page No	:	105
Abstract	:	NQT-SHE cells derived from Syrian hamster embryo (SHE) fibroblasts transformed by 4-nitroquinoline-1-oxide synthesize two modified usd\alphausd2(I) subunits of type I collagen designated as N-33 and N-50 (N-collagens). The N-collagens migrate more slowly than the normal usd\alphausd2(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 usd\alphausd2(I) chain and in two peptides (V8-1 and V8-2) obtained by V8 protease digestion of the modified usd\alphausd2(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 N33usd\alphausd and N50usd\alphausd chains with linkage-specific neuraminidase showed that acidicity was due to the presence of sialic acid in an usd\alpha 2\to 6usd 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 usd\alphausd2(I) chain during SDS-PAGE. The usd\alphausd chains and purified CB3,5 peptides from the N-collagens reacted with biotinylated Jacalin lectin providing additional evidence for O-glycosylation. Secretion of a prousd\alphausd2(I) monomer is unexpected since the usd\lbrack\alpha 2(I)\rbrack\sb3usd 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 N33usd\alphausd and N50usd\alphausd 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 usd\alphausd2(I) chain occurs in the cis-Golgi while galactose and sialic acid may be added in the trans-Golgi network.
Subject	:	Biochemistry
	:	Biological sciences
	:	collagen
	:	fibroblasts
	:	Pure sciences
	:	Syrian hamster embryo