Expression and molecular characterization of an intriguing hyaluronan synthase (HAS) from the symbiont “Candidatus Mycoplasma liparidae” in snailfish

Plasmid construction

To obtain the exogenously expressed protein snHAS, we inserted the snHAS gene sequence into an exogenous expression vector. The genomic DNA extraction, sequencing, assembly, and annotation were performed according to our previously published paper (Lian et al., 2020). In brief, a genomic DNA library of the hadal snailfish gut was constructed. Based on the sequences of the annotated HAS, two pairs of primers were designed, which were listed in Table S1. The HAS gene was amplified using PCR with the following protocol: a 50 µl reaction mixture was prepared containing five ng of the previously prepared cDNA, four µl of 10 mM dNTPs, 10 µl 5 × PCR buffer, 0.5 µl Primer STAR HS DNA Polymerase (Takara, Tokyo, Japan), two µl of 10 µM for each primer, and 32.5 µl of ddH₂O. The PCR program was as follows: 98 °C for 10 s for denaturation, 30 cycles of 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. A template of full-length snHAS was used when the E173D mutant was generated with primers listed in Table S1. A pET-28a(+) vector was ligated with the purified PCR product. Sequencing (BGI, Shenzhen, China) was used to identify the positive clones, and subsequently, the expression of the target gene was carried out in BL21 (DE3) competent cells.

Expression and purification

The wildtype and mutant of His-snHAS were expressed in BL21 (DE3) cells. Purification of His-snHAS wildtype and mutant was performed following the manufacturer’ protocol (Sangon, Shanghai, China). In summary, a media containing 50 µg/ml of LB was used to culture the E. coli BL21 (DE3), which has recombinant plasmid, at 37 °C for 8 h. LB broth was used to dilute the cultures with 1,000:1 and the cultures were subjected to further incubation. Once the OD₆₀₀ value of the cultures reached approximately 0.6, a final concentration of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma, Shanghai, China) was added to induce the expression of target gene. Cells were collected after induction for 16 h at 28 °C and were then lysed using an ultrasonicator in a binding buffer (20 mM Tris–HCl, 10 mM imidazole, 500 mM NaCl) kept on ice. After ultrasonication, cell debris was removed by centrifugation at 8,000 ×g for 35 mins at 4 °C. The supernatant was retained and mixed with Ni-NTA 6FF Sefinose Resin on ice. Recombinant proteins were eluted after thorough washing with a washing buffer (500 mM NaCl, 10 mM imidazole, 20 mM Tris–HCl). 1 ×PBS was used to dialyze the purified proteins at 4 °C for 24 h with buffer replacement every 12 h, carried out three times in total. The Bradford method was employed to determine the protein concentration, using bovine serum albumin (BSA) as the standard. The purified proteins were combined with a 6 ×SDS gel-loading buffer. After boiling for 10 min at 100 °C, the mixture was resolved with 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Coomassie brilliant blue R250 was used to stain the gels. Finally, the purified proteins were stored in aliquots at −80 °C or used immediately, unless specified otherwise.

Hyaluronan synthase assay

The activity of snHAS and its mutant snHAS E173D was examined using the following procedures. The assay was performed in a 50 µl reaction containing 15 mM MgCl₂, 50 mM phosphate buffer saline (PBS), five mM DL-dithiothreitol (DTT), one mM UDP-GlcNAc, one mM UDP-GlcA, and 30 µM purified snHAS wildtype or its mutant. After incubating the mixture for 1 h at 37 °C, the reaction was stopped by boiling, and then the reaction was tested by an HA Detection Kit following the manufacture’s protocol (Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai, China). Reactions without HAS were used as the control. In brief, HA binding protein (HABP) and horse radish peroxidase (HRP) were applied as primary and secondary probes, respectively. After chromogenic reaction with TMB, the OD₄₅₀ was recorded using Varioskan LUX (Thermo Fisher Scientific, Waltham, MA, USA). Each assay was repeated at least three times in this study. One enzymatic unit was defined on the amount of enzyme required when one pmol of HA in 1 min was synthesized within the above reaction.

For the hyaluronidase treatment, the synthesis reaction was monitored at 5 mins intervals for the first 40 mins, followed by 10 mins intervals for another 20 mins. After this, the mixture was incubated with two µg/µl hyaluronidase at 37 °C for 1 h, and assessed at 5 mins intervals. The reaction without hyaluronidase was used as a control.

Enzyme kinetic analysis

The K_m values of snHAS and its mutant were determined by measuring enzyme activities in the presence of UDP-GlcNAc and UDP-GlcA at various concentrations. For the wildtype snHAS, 30 µM of studied enzyme protein was used, with UDP-GlcA at a fixed concentration of one mM, and UDP-GlcNAc at varied concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mM) were used. Alternatively, UDP-GlcNAc was at a fixed concentration of one mM, and UDP-GlcA at varied concentrations (0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 mM) were used. For the mutant snHAS E173D, 30 µM of the enzyme, UDP-GlcA at a fixed concentration of one mM, and UDP-GlcNAc at varied concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mM) were used. Alternatively, UDP-GlcNAc at a fixed concentration of one mM, and UDP-GlcA at varied concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mM) were used. A nonlinear least-square fitting procedure was employed to calculate the kinetic parameters of K_m and V_max, using the Michaelis–Menten equation and curve fitting software.

In the experiment assessing the effect of cardiolipin on snHAS, freshly prepared proteins were used in the reaction mixture (15 mM MgCl₂, 50 mM PBS, five mM DTT, one mM UDP-GlcNAc, one mM UDP-GlcA, 30 µM purified snHAS) along with various concentrations of cardiolipin (1.0, 2.0, 3.0 mM). The reaction was incubated for 1 h at 37 °C, and the activities were detected using the methods described above.

The molecular weight (MW) of HA

After the synthesis reaction was completed, three volumes of ethanol was added to the reaction. The mixture was then stored for 1 h at 4 °C. Following this, it was subjected to centrifugation at 5,000 r/min for 15 mins at 4 °C. The supernatant was then washed with an equal volume ethanol salt solution (75% w/v ethanol and 25% w/v 0.2 M NaCl). After another round of centrifugation at 10,000 rpm/min for 15 mins at 4 °C, the supernatant was discarded, and the sediment was freeze-dried. The purified HA was used for molecular weight detection. Briefly, the purified HA (5 mg/ml) was prepared along with standards (5000-667800 Da; Sigma-Aldrich, St. Louis, MO, USA) and analyzed using High Performance Gel Permeation Chromatography (HPGPC) with a BRT105-104-102 connected gel column (8 ×300 mm) (BoRui Saccharide Biotech Co. Ltd, Shenzhen, China), A 0.05 M NaCl was utilized as mobile phase, and the flow rate was set to 0.6 ml/min. The detection was carried out using an RI-10A refractive index detector.

Characteristics of snHAS sequence

The gene encoding snHAS was isolated from the cDNA library of the symbiotic “Candidatus Mycoplasma liparidae” residing in the hadal snailfish. The snHAS gene spans 933 base pairs, encoding a protein with 310 amino acids (accession number WWS21520) with a molecular weight (MW) of 37 kDa. A comparative analysis of snHAS with other HASs from diverse organisms was conducted (Fig. 1). Results revealed that compared with the motif of Class I HASs typically composed of D, D, QXXRW (Kooy et al., 2013; DeAngelis & Zimmer, 2023), only two residues, 88D and 208Q, were conserved in snHAS (Fig. 1A). In contrast to Class I HASs, Class II HAS typically contains two GT2 modules (Table 1), whereas snHAS harbors only one predicted GT2 module (Fig. 1). Additionally, snHAS exhibits two motifs, DGSTD (38-42aa) and DX DD (88-91aa), akin to those found in the second GT2 module of Class II HASs (Fig. 1B). Furthermore, while 2-6 “B(X ₇)B” HA binding motifs were predicted in HASs from Mycoplasma and other organisms, only one such motif (273-281aa) was identified in snHAS (Table 1). Notably, snHAS lacks any predicted transmembrane regions, differing from glycosyltransferases in other Mycoplasma species (0-1) and HASs in other organisms (2-7), including both microorganisms and multicellular organisms (Table 1). Moreover, snHAS exhibits the smallest MW of 37.2 kDa, contrasting with the MWs of other previous reported HASs, which range from 44.78 to 112 kDa (Table 1). The isoelectric point (pI) values of these HASs range from 7.28 to 9.08, with pmHAS displaying the lowest value of 7.28. Notably, no signal peptides were predicted in any of these HASs, including snHAS.

Phylogenetic analysis of snHAS

To better understand the evolutionary relationship of snHAS with the glycosyltransferases from other organisms, a phylogenetic tree was constructed using sequences selected from the NCBI database. The results revealed that snHAS clustered closely withglycosyltransferases from the Mycoplasmatota bacterium (MDK289671, MDR2568307, MDR1235299), all of which belong to the Mycoplasmataceae family (Fig. 2). The sequence similarities to snHAS were 47.08%, 41.78% and 31.33% at the amino acid level. HASs of Class I were grouped together. The HASs from Mycoplasma formed a distinct cluster (Fig. 2).

Catalytic activity

The catalytic activity of snHAS was examined using UDP-GlcNAc and UDP-GlcA as substrates. The results, presented in Fig. S1, demonstrated that snHAS could synthesize HA, with the amount of product positively correlated with the substrate concentrations prior to saturation. To verify the identity of the synthesized product, hyaluronidase was applied. The results showed that after treatment with hyaluronidase, the amount of HA decreased rapidly to almost undetectable levels (Fig. 3), confirming that the product synthesized was indeed hyaluronic acid. Subsequently, the enzymatic characteristics of snHAS were investigated. The results, displayed in Table 2, indicated that the K_m value of snHAS for UDP-GlcNAc was 0.26 ± 0.05 mM, which was significantly lower than that of HAS from other organisms (Table 2). Meanwhile, the K_m for UDP-GlcA was 0.04 ± 0.01 mM, comparable to enzymes from other sources (Table 2). For the k_cat/K_m values, snHAS exhibited a higher value for UDP-GlcNAc compared to spHAS and a similar value to seHAS. Conversely, the k_cat/K_m value of snHAS for UDP-GlcA was slightly lower than that of spHAS but comparable to seHAS (Table 2). The conserved motif D, D, QXXRW in class I HAS was partially conserved in snHAS, with only the first D and Q being conserved. To investigate whether the residue “173D” is critical for the acticity of snHAS, we introduced a mutation that changed the amino acid E at the second D position to D, referred to as snHAS E173D mutant. The snHAS E173D mutant was cloned and overexpressed in vitro. Its activity was then examined and compared to that of the wild-type snHAS. The results showed that the snHAS E173D mutant retained HA synthesis activity. The K_m values of snHAS E173D were 0.21 ± 0.02 mM for UDP-GlcA and 0.41 ± 0.07 mM for UDP-GlcNAc, representing increases of 1.26- and 5.25-fold over the wild-type, respectively. Additionally, the k_cat/K_m value for the E173D mutant with UDP-GlcNAc was 5.9-fold higher than that of the wild-type, while the k_cat/K_m value for UDP-GlcA was lower than that of the wild-type (Table 2).

The effect of cardolipin

To explore the effect of cardiolipin on the activity of snHAS, cardiolipin was added to the reaction at final concentrations of one mM, two mM, and three mM. The results indicated that the activity of snHAS did not show significant changes when cardiolipin was present compared to the reaction that did not include cardiolipin (Fig. 4).

Measurement of HA molecular weight

To investigate the molecular weight of HA synthesized by snHAS, the molecular weight was examined by HPGPC. Results showed two distinct peaks. Upon comparison with the control, the molecular weight of HA synthesized by snHAS was approximately 3.5 kDa and 8.4 kDa (Fig. 5).