Figure 4
Phosphacan/PTPRZ is identified as a GlcNAc6ST3-modifying KS/CSPG expressed in the cerebral cortex of adult mice. (a) R-10G immunoprecipitated proteins (MW > 300 kDa) prepared from the cerebral cortex of adult WT, GlcNAc6ST2-KO, and GlcNAc6ST3-KO mice were subjected to an LC-MS/MS analysis (Fig. S7). Positions and sequences of the four identified peptides in the samples of WT and GlcNAc6ST2-KO mice, but not GlcNAc6ST3-KO mice, corresponding to phosphacan/PTPRZ are indicated. Three major isoforms of phosphacan/PTPRZ expressed in the brain are depicted. Positions of the CS GAG modification sites (open arrowheads) and those of the O-mannose modifications (closed arrowheads) are indicated50,51. The short splicing isoform lacks the peptide stretch of the PTPRZ full-length (PTPRZ-FL), which contains the sites of CS and O-mannose modifications. Phosphacan is the secreted isoform of PTPRZ lacking transmembrane domain (TM) and the dual phosphatase domains (PTPc). A consensus carbonic anhydrase alpha-related domain (CAH) and a fibronectin type III domain (FN) are shared by these three variants. Positions of nine potential N-linked glycosylation sites and those of several O-GalNAc modifications51 are not indicated. (b) The samples of 1% Triton-soluble fractions prepared from adult WT cortex were immunoprecipitated with R-10G. R-10G-bound and R-10G-unbound materials were immunoblotted and probed with either Ptprz-S34 or R-10G antibody. (c) The RNA-seq transcriptome results of the gene of phosphacan/PTPRZ in indicated cell types are shown49. (d) The samples of 1% Triton-soluble fractions prepared from the cerebral cortex of adult WT mice and GlcNAc6ST1, 2, 3, and 4 KO mice were immunoprecipitated with R-10G. The R-10G-bound materials were immunoblotted and probed with the Ptprz-S antibody. Representative results are shown (n = 2 in b,d). Full-length blot images are presented in Supplementary Fig. S8.
