Sulfation pattern of chondroitin sulfates regulates SHP2, the non‐receptor tyrosine phosphatase
Authors: Tobacman Joanne Kramer , Bhattacharyya Sumit , Feferman Leo 
Published: November 2023

In this study, the interaction between the chondroitin sulfation patterns of chondroitin 4‐sulfate (C4S) and chondroitin 6‐sulfate (C6S) and SHP2 (tyrosine‐protein phosphatase non‐receptor type 11 (PTPN11), also known as protein‐tyrosine phosphatase 1D (PTP‐1D) or PTP‐2C) was examined. SHP2 is a universal and critical tyrosine phosphatase, linked to over 50 vital biological processes and shown to interact with over 30 specific phosphorylations. SHP2 has two tandem Src homology‐2 domains enabling tyrosine phosphatase function. Previously, we showed in HepG2 cells that SHP2 was sequestered when chondroitin 4‐sulfation was increased due to decline in Arylsulfatase B (ARSB; N‐acetylgalactosamine 4‐sulfatase). SHP2 activity was reduced, leading to increase in the downstream phosphorylation of p38‐MAPK. In human prostate stem cells, the chondroitin sulfation pattern was modulated either by silencing with siRNA or overexpressing by transfection with specific plasmid of two sulfatase enzymes, ARSB or N‐acetylgalactosamine 6‐sulfatase (GALNS). Silencing decreased ARSB or GALNS activity to less than 12% of the baseline level, and increased the total sulfated glycosaminoglycan content significantly in the human prostate stem cells. ARSB silencing induced a 74% increase of C4S above the basal level, and GALNS silencing increased the C6S to almost two times the control level. The C4S/C6S ratio increased from 1.58 to 2.57 after ARSB silencing and decreased to 0.82 after GALNS silencing. In contrast, ARSB overexpression reduced the C4S content and GALNS overexpression reduced the C6S content. The C4S/C6S ratio declined after ARSB overexpression and increased after GALNS overexpression. Both ARSB silencing and GALNS overexpression significantly (p<0.001) inhibited the SHP2 activity. In contrast, ARSB overexpression and GALNS silencing increased the SHP2 activity significantly (p<0.001) above the baseline level. To further examine the phosphatase‐chondroitin sulfate interaction, SHP2 binding to three different chondroitin sulfates was compared. The sulfatases included chondroitin 6‐sulfate from shark cartilage (>90% C6S), chondroitin 4‐sulfate from sturgeon notochord (>90% C4S), and chondroitin 4‐sulfate from bovine trachea (~70% C4S). These chondroitin sulfates were biotinylated with EZ link ‐ Hydrazide‐PEG4‐ Biotin. The binding of GST‐labeled SHP2 with the biotinylated chondroitin sulfates (CS) was measured using PTPN11‐GST and microplates coated with GST antibody. Maximum binding occurred with C4S, in preference to binding with C6S or a mixture of C4S and C6S (70% 4S, 30% 6S). Displacement studies further demonstrated that chondroitin sulfates with higher 4‐sulfate content had much higher affinity to bind with SHP2. C6S had a very low affinity and interfered with the binding of C4S with SHP2. These data collectively indicate that when chondroitin 4‐sulfation of GAGs increases or there is a relative increase in C4S, the binding with SHP2 increases, and the SHP2 phosphatase activity is reduced. Thus, the regulation of multiple downstream tyrosine phosphorylations can be affected by sustained phosphorylation following increase in C4S. The prolonged activation of phosphate‐mediated signaling and transcriptional events follow the changes in sulfatases and the associated changes in chondroitin sulfation.