Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X₇-B motif

Abstract

Hyaluronan synthases (HAS) normally make large (>MDa) hyaluronan (HA) products. Smaller HA fragments (e.g. 100-400 kDa) produced in vivo are associated with inflammation and cell signaling by HA receptors that bind small, but not large, HA. Although HA fragments can arise from breakdown by hyaluronidases, HAS might also be regulated directly to synthesize small HA. Here we examined the Streptococcus equisimilis HAS (SeHAS) C-terminus, which contains a tandem B-X 7 -B motif (K 398 -X 7 -R 406 -X 7 -K 414 ), by testing the effects of 27 site-specific scanning mutations and 7 C-terminal truncations on HA synthesis activity and weight-average mass. Although HAS enzymes cannot be HA-binding proteins, these motifs are highly conserved within the Class I HAS family. Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines. Glycobiology 22:1302-1310] that HAS uses two independent activities to control HA size and HA synthesis rate; these are two separate functions. We conclude that HAS regulatory modifications that alter tandem B-X 7 -B motif conformation could mimic these mutagenesis-induced effects, allowing HAS in vivo to make small HA directly. The results also support a model in which the tandem-motif region is part of the intra-HAS pore and interacts directly with HA.

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1

“…In physiological conditions, HAS1 and HAS2 synthesize HA polymers of high molecular mass, while HAS3 synthesizes shorter HA polymers (24), and the production of low molecular mass HA can be obtained by the action of degrading enzymes as well as by oxidative stress or ultraviolet light (25,26). Interestingly, the stoichiometry of the cytosolic UDP substrates has a critical role to define HA polymer length, and the C-terminal region of HASes appears to have regulatory functions (27).…”

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

“…Similarly, it has been shown that the catalytic activity of β‐1,4‐galactosyltransferase is modulated by the conformational change brought forth by its two flexible loops . Interestingly, T40L is a part of the HAS tandem‐motif region described to be involved in both the control of HA synthesis rate and the control of HA size for class I seHAS . Furthermore, MD simulations show that the increase in flexibility caused by the substitutions results in the N‐terminal region first interacting with the GlcNAc‐transferase and then turning towards the GlcA‐transferase domain of pmHAS (Figures B and S8).…”

Identification and Comparison of Hyaluronic Acid Biosynthetic Genes from Different Capsular Types of Pasteurella Multocida

“…The protein structure of GlmU was previously studied in E. coli, and the catalytic site of this protein was also matched to the variable binding regions predicted from the P. multocida strains in this study. These variable regions correlated with N-terminal pyrophosphorylase and C-terminal acetyltransferase catalytic sites [39,40]. GlmU has two active sites: (i) acetyltransferase, which is responsible for CoA-dependent acetylation of D-glucosamine-1-P to N-acetyl-D-glucosamine-1-P at the C-terminal domain, and (ii) pyrophosphorylase, which catalyses the transfer of uridyl from UTP to D-glucosamine-1-P, forming UDP-N-acetyl-D-glucosamine and pyrophosphate at the N-terminal domain [40].…”