Fig. 1.

Mutant Fam20b proteins have severely hypomorphic kinase activities. (A) Model illustrating that Fam20b transiently phosphorylates xylose, the first sugar added to a serine residue of the core protein during synthesis of a chondroitin sulfate PG (Koike et al., 2009). 2-Phosphoxylose phosphatase (PXYLP) removes the phosphate, thus promoting efficient glycosaminoglycan side chain outgrowth (Koike et al., 2014). (B) Culture medium from COS-1 cells transfected with secreted forms of zebrafish Fam20b or vector alone was incubated with IgG-Sepharose, and proteins purified from the medium were subjected to SDS-PAGE. Expression of each protein A-tagged protein was examined with anti-mouse IgG antibody. Lane 1: Fam20b^wt; lane 2: Fam20b^b1125; lane 3: Fam20b^b1127; lane 4: protein marker; lane 5: vector alone. (C) After normalization from immunoblots in B, evaluation of kinase activity demonstrated that the mutant kinases Fam20b^b1125 and Fam20b^b1127 both have a significant decrease in activity compared with Fam20b^wt, with Fam20b^b1127 displaying significantly less activity than Fam20b^b1125. Both mutant kinases, however, showed more activity than the negative, empty-vector control. Data are mean+s.e.m. (n=3, Tukey's multiple comparison test, *P<0.05). CS, chondroitin sulfate; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcUA, glucuronic acid; P, phosphate; Ser, serine; Xyl, xylose.