New insights into the alternating sequences of heparan sulfate

  The sulfated domains (S‐domains) within HS are thought to be the primary binding site for protein ligands (Gallagher and Lyon, 2000). Studies on the binding of multimeric chemokines to HS have suggested that N ‐acetylated sequences (NAc‐domains) act to space the S‐domains correctly so binding can occur at more than one HS binding site within a protein (Lortat‐Jacob et al . 1995; Stringer et al . 2002). So far, little functional significance has been ascribed to either alternating N ‐sulfate/NAc or lone N ‐sulfate types of sequence, although alternating sequences are thought to occur at the borders between S‐domains and NAc‐domains, forming transition zones between the two. Existing scission techniques used to excise oligosacchrides from the intact chain for structural and functional analysis disrupt the transition zones (heparinase III), or the S‐domains (heparinase I) or both (low pH nitrous acid). Materials and methods  Radiolabelled HS was isolated from cultured 3T3 fibroblasts HS. Its patterns of degradation using bacteriophage K5 lyase as well as conventional heparinases I/III and nitrous acid were compared. K5 lyase‐resistant fragments were characterized by size exclusion chromatography and strong‐anion exchange HPLC. Results  The work presented demonstrates how the enzyme K5‐lyase specifically cleaves only within NAc‐domains requiring sequences of at least five consecutive N ‐acetylated disaccharides within the HS chain. The enzyme leaves intact S‐domains together with their flanking transition zones in situ . Discussion  The characterization of oligosaccharides generated by K5 lyase cleavage of the intact HS chain provides new insights into the domain structure of HS.