Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life

Abstract

Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.

Keywords: hyaluronan; in vitro reconstitution; membrane transport; oligomeric state; polysaccharide.

Fig. 1.

Hyaluronan synthases’s catalytic activity is sensitive to delipidation. (A) Catalytic activity of Se-HAS in inverted membrane vesicles (IMVs) and detergent-solubilized states. Inset: Anti-His western blot of samples used for activity assays. (−): Control reaction in the absence of UDP-GA. (B) Same as (A) but for Cv-HAS. L: LDAO, C: sodium cholate, D: DDM, T: Triton X-100, Dig: digitonin, LF: LFCE14, DPM: disintegrations per minute. “-r” indicates samples solubilized in the respective detergents and re-reconstituted directly after solubilization. Full-length Cv-HAS is indicated with an arrow. Error bars are the standard deviations of the means from three independent experiments.

Fig. 2.

Oligomerization of bacterial HAS as biologically functional unit. (A) Co-purification of co-expressed His- and FLAG-tagged Se-HAS using Ni-NTA (left) and FLAG-agarose (right), shown as western blots against the affinity tags. Sol: Detergent-solubilized material; W, E: Wash and elution buffers. (B) Tandem purification of His- and FLAG-tagged Se-HAS. The material eluted from Ni-NTA was purified using FLAG-agarose. (C) Catalytic activity of tandem purified Se-HAS. DPM: disintegrations per minute, Error bars represent deviations from the means from at least three independent replicas. (D) As (A) but for co-expressed His-tagged wild type (WT) and catalytically inactive (D260N) FLAG-tagged Se-HAS. (E) As (D) but tandem purification of co-expressed WT and D260N Se-HAS. Left panel: Illustration of purification steps. Black and white circles indicate His- and FLAG-tagged WT and D260N Se-HAS, respectively. 1./2. Material recovered after Ni-NTA and FLAG-agarose purification, respectively. (F) Catalytic activity of tandem purified WT and D260N Se-HAS. HA biosynthetic activity was assessed after the first (Ni-NTA) and second (FLAG-agarose) affinity purification. Error bars as in (C). (G) As (D) but tandem purification of His- and FLAG-tagged D260N Se-HAS. (H) Catalytically inactive Se-HAS inhibits activity of WT Se-HAS. Individually purified SNAP-tagged WT and D260N Se-HAS were combined at the indicated molar ratios (WT:D260N) and reconstituted into lipid vesicles. Catalytic activity is expressed relative to the activity in the absence of D260N. Inset: western blot of purified SNAP-tagged WT and D260N Se-HAS. Error bars as in (C). (I) Purification of His- and FLAG-tagged Cv-HAS using FLAG-agarose. Additional bands observed in the ‘E’ fraction on the α-His blot represent cross-reactive contaminants shedding from the anti-FLAG-agarose beads. (J) Catalytic activity of FLAG-agarose purified Cv-HAS. Error bars as in (C). All experiments were performed with digitonin-solubilized protein.

Fig. 3.

Se- and Cv-HAS adopt different oligomeric states in biological membranes. (A) Representative fluorescently-labeled Se-HAS particles in a supported lipid bilayer with bleaching profile in a TIRF field. Scale bar: 10 μm. (B) Bleaching step distribution of 1181 and 968 Se-HAS particles in the presence and absence of the UDP-GA substrate, respectively. Error bars represent the deviations from the mean from at least three different reconstitutions. (C) Bleaching step distribution of 820 Se-HAS particles after co-reconstituting equimolar ratios of fluorescently-labeled and unlabeled Se-HAS. Error bars as in (B). (D) As in (B) but for 770 and 795 particles of catalytically inactive Se-HAS (D260N) in the presence and absence of the UDP-GA substrate, respectively. (E) Representative bleaching profile of fluorescently labeled Cv-HAS. (F) Distribution of bleaching steps for 713 and 830 particles of Cv-HAS in the presence and absence of UDP-GA substrate, respectively. Error bars as in (B).

Fig. 4.

Quaternary structures of Se- and Cv-HAS during HA biosynthesis. (A) Experimental outline of the supported bilayer containing AlexaFluor647-labeled HAS and HA detection via biotinylated HA-binding protein (HABP) and AlexaFluor546-conjugated streptavidin. (B) Total AlexaFluor546 fluorescence intensity of the supported bilayer containing the indicated Se-HAS to lipid molar ratios after HA labeling. No background labeling is observed in the absence of HABP or for the catalytically inactive D260N mutant. (C) Bleaching step distribution of WT and D260N Se-HAS particles (black and gray columns, respectively) and co-localization with HA. Green columns represent the fraction of each Se-HAS population that co-localizes with the HA signal. Steps: Bleaching steps observed in the 647 nm channel. Inset: number of particles measured. (D) Same as in (C) for WT Se-HAS and HA-labeling in the absence of HABP. (E) and (F) As (C and D) but for AlexaFluor647-labeled Cv-HAS. (C–F) The data shown is from at least four reconstituted bilayers from at least two independent protein preparations.

Fig. 5.

Site-specific cross-linking of Se-HAS. (A) Inverted membrane vesicles containing Se-HAS carrying the UV-inducible cross-linker para-benzoylphenylalanine (Bpa) at the indicated positions were exposed to UV light and analyzed by anti-FLAG western blotting. Monomeric and dimeric Se-HAS species are labeled ‘M’ and ‘D’, respectively. (B) The indicated Se-HAS Bpa-mutants were purified, reconstituted and exposed to UV light and analyzed by western blotting. −/+: without and with UV exposure. The asterisk indicates a nonspecific high molecular weight band occasionally observed for WT Se-HAS. (C) Predicted topology of Se-HAS. Stars indicated positions where cross-linking was observed.

Fig. 6.

Comparison of HA biosynthesis by different HAS. (A) Distributions of in vitro synthesized HA after ¹⁴C-GA labeling, electrophoresis and autoradiography. (+/−): Synthesis in the presence of both (UDP-GA/-GlcNAc) and only one (UDP-GA) substrate, respectively. D: Degradation with hyaluronidase, E: UDP-¹⁴C-GA tracer added after termination of HA biosynthesis with EDTA. (B) UDP-labeling of in vitro synthesized HA. HA was synthesized with unlabeled substrates followed by addition of either UDP-¹⁴C-GA (¹⁴C-GA) or ¹⁴C-UDP-GA (¹⁴C-U) prior to electrophoresis and autoradiography. Right panel: ¹⁴C-labeled HA generated by Se-HAS was degraded with hyaluronidase for 0–45 min. (C) Chemical cleavage of the ¹⁴C-label on Se-HAS-produced HA upon incubation in 0.1 N sodium hydroxide.

Fig. 7.

Model of HA biosynthesis by dimeric and monomeric HASs. Dimeric bacterial HAS forms a single HA TM channel that is associated with two GT domains. Elongation at the reducing end generates a UDP-linked polymer bound to one GT domain. The last incorporated sugar modulates the selectivity of the second GT domain, thereby ensuring alternating incorporation of GA and GlcNAc. Following glycosyl transfer, the nascent HA retains a UDP moiety bound at the second GT domain. Concomitant to glycosyl transfer, the polymer moves into the TM channel by one sugar unit. Monomeric viral HAS elongates the non-reducing end of HA and the single GT domain binds UDP-activated substrates to trigger glycosyl transfer. Subsequently, UDP is released to allow binding of a new substrate molecule.