The intracellular domain of Hyaluronan synthase can produce monodisperse low- molecular weight hyaluronic acid with functional activity

Abstract Background: The biological properties of hyaluronan (HA) are determined by its molecular weight (MW). Most natural extraction process of HA resulted in a polydisperse polymer. In the present study, the monodisperse LMW-HA was biosynthesized by truncated recombinant hyaluronan synthase (HAS) derived from Streptococcus equisimilis (SeHAS). The effects of t transmembrane domains truncation of SeHAS on productivity and specific activity of enzymes, dispersity, MW, and biological activity of synthesized polymers were investigated. The recombinant HAS 123 (341 aa), HAS 23 (309 aa), and HAS Intra (263 aa) were constructed. The MW, structure, and titer of the polymer were studied by electrophoresis, FTIR spectroscopy and carbazole assay, respectively. The biological activity of HA polymers was assessed by cell proliferation and wound healing assays. Results: The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 mg HA .mg protein -1 .min -1 for SeHAS, HAS 123 , HAS 23 , and HAS Intra , respectively. The results revealed SeHAS produced a polydispersed HMW-polymer (268 kDa), while HAS 123 and HAS 23 produced a polydisperse LMW-polymer (6.1, and 4.6 kDa, respectively). Interestingly, HAS Intra produced a monodisperse LMW-HA (1.9 kDa). Biological assessments indicated the LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest wound healing effect at 10 µg/mL, while by increasing in the concentration to 200 µg/mL, both LMW- and HMW-HAs postponed the wound healing recovery rate. Conclusion: The study indicated the transmembrane domains of SeHAS affect the titer, size, and dispersity of synthesized HA. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.