Establishment of a method for measuring the 3- <i>O</i> -sulfated structure in heparan sulfate

Heparan sulfate is present on the cell surface and within the extracellular matrix of most animal species, and it regulates various physiological processes by binding to a wide variety of functional proteins. The diverse array of sulfation patterns on the heparan sulfate chain enables specific binding to each protein. Of particular interest is the 3-O-sulfated (3S) structure of glucosamine residues, which is considered a key structure in determining binding specificity to functional proteins. However, 3-O-sulfation is a rare modification, which makes it difficult to analyze and hinders elucidation of its physiological function. Establishing a general method for measuring the 3S structure is thus important for continued progress in this research field. We previously developed an HPLC method to separate and quantify 13 components of heparan sulfate, including five 3S components, as a complete heparin lyase digestion product. Application of this method in routine analysis required the development of a standard sample for quantitation that is simple to prepare in large amounts and exhibits good stability. A heparan sulfate standard composed of 13 components in known quantities, designated HS13, was prepared for this purpose. A standard mixture of the 13 components can be obtained by digesting HS13 with a heparin lyase. A compositional analysis of heparan sulfate derived from various rat organs was then conducted to test the newly developed standard. The organ-specific distribution of each 3S component was elucidated, and it was confirmed that the 3S components in biological samples can be quantified by routine HPLC analysis.