title: Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase.
abstract: Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability.
authors.affiliation: From the Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Baumann
authors.affiliation: From the Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland thierry.hennet@uzh.ch.
authors.forename: Thierry
authors.identifier: 0000-0002-7276-737X
authors.initials: T
authors.lastname: Hennet
mesh_terms: D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000235:genetics; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding
chemical_list: D009363:Neoplasm Proteins; D003094:Collagen; C535154:COLGALT2 protein, human; D005700:Galactosyltransferases; C535153:COLGALT1 protein, human
keywords: CRISPR/Cas; collagen; endoplasmic reticulum (ER); glycosylation; osteosarcoma; protein trafficking (Golgi)