Structural snapshots of hyaluronan formation reveal principles of length control and secretion

Abstract

Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) units reaching several megadaltons in healthy tissues. HA is synthesized and secreted in a coupled reaction by HA-synthase (HAS). Here, structural snapshots of HAS provide important insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We reveal a loop insertion mechanism for substrate binding, monitor the extension of a GlcNAc primer with GlcA, and capture the opening of a secretion channel that coordinates a nascent HA polymer. Further, we identify HA-interacting residues that control HA product lengths. Integrating structural and biochemical analyses, we propose a mechanism for HA length control based on finely tuned enzymatic processivity and catalytic rates.

Molecular insights into capsular polysaccharide secretion

“…The CPS was eluted from the column after a 30-min incubation with 2 M NaCl, 100 mM sodium citrate pH 3.0 and 0.01 % LMNG, concentrated to 250 μl using a 3-kDa filter (Amicon) and dialysed overnight against PBS in a 3.5-kDa dialysis membrane. The next day, the sample was run on a 1.5% agarose gel (Ultra-pure agarose, Invitrogen) or a 4-20% gradient polyacrylamide gel (Bio-Rad), and stained with Stains-All dye (Sigma) as described 49 . The obtained CPS sample was also used for cryo-EM analyses.…”