Recent analytical advances in the detection and characterization of 3-O-sulfated heparan sulfate.

Heparan sulfate (HS) is a linear, highly sulfated, and heterogeneous polysaccharide that covalently attaches to core proteins to form heparan sulfate proteoglycans (HSPGs). HSPGs are widely expressed in mammalian cells and are found on the cell surface and within the extracellular matrix (ECM). Structurally, HS consists of repeating disaccharide units composed of hexuronic acid (HexA) (either glucuronic acid (GlcA) or iduronic acid (IdoA)) linked to glucosamine (GlcN) units. The HS chain undergoes extensive post-polymerization modifications, including N-deacetylation of GlcN, C5-epimerization of HexA, and sulfation at various positions like 2-O-sulfation of HexA, as well as 3-O-, 6-O-, and N-sulfation of GlcN. Among these modifications, 3-O-sulfation of HS, produced by HS 3-O-sulfotransferase (HS3OST), is the rarest and most functionally significant. While 3-O-sulfated HS is well known for its anticoagulant properties through the activation of antithrombin, it also plays a critical role in various physiological and pathological processes, including cell differentiation, cancer progression, herpes simplex virus entry, and neuronal development. However, the precise mechanisms underlying these functions and their pathological implications remain inadequately characterized. This knowledge gap is primarily due to the low abundance of 3-O-sulfated HS and the lack of standardized analytical methods for its detection in biological samples. In this review, we summarize recent advancements in analytical techniques for the analysis of 3-O-sulfated HS and highlight potential future directions to improve its characterization and advance our understanding of its biological roles.