Structural Domain Analysis of Heparin and Heparan Sulfate Combined With Label-Free Quantitative Proteomics to Elucidate Their Functional Diversity in Liver Cancer and APAP-Induced Liver Injury

The precise structure of glycosaminoglycans is critical for their bioactivity and the development of glycopharmaceuticals. Herein, cellular and animal experiments were conducted to assess the differences in the activities of heparin (HP) and heparan sulfate (HS) against liver cancer and drug-induced liver injury. Label-free quantitative proteomics, bioinformatics, biolayer interferometry, and immunohistochemical analyses were used to determine key proteins with differential expression. As a result, HP demonstrated superior antiliver cancer activity compared with HS, whereas HS exhibited strong potential in resisting acetaminophen-induced liver injury. DIRAS family GTPase 2 (DIRAS2) was identified as a key HS-binding protein that was strongly associated with cell proliferation, and its expression levels in cells and tissues showed opposite trends following HP and HS administration. HP significantly reduced the abundance of DIRAS2 in the tumor tissue, thereby inhibiting tumor cell proliferation, whereas HS promoted proliferation by increasing DIRAS2 expression. Cluster sequencing revealed that consecutive GlcNS6S-IdoA2S domains in HP and IdoA2S-GlcNS6S, GlcA-GlcNS6S, and IdoA-GlcNAc domains in HS were required for affinity binding within the decasaccharide region. Molecular docking suggested that differences in the binding modes of HP and HS chains to DIRAS2 underlie their functional diversity. These findings indicate that HP and HS oligosaccharides with well-defined structures may serve as potential therapeutic agents for liver-related diseases.