Lysosomal hexosaminidases are one of the widely studied and crucial glycosidases that are structurally and evolutionarily conserved among vertebrates and have also been shown to be conserved in some invertebrates across the animal kingdom. Their role in lysosomal degradation of glycoconjugates and in chitin depolymerization in several organisms is of great physiological significance, and hexosaminidases are proven to be versatile biosynthetic tools [35, 36]. Owing to their functional importance and clinical significance, hexosaminidases have been purified, characterized, and studied from diverse group of organisms – especially from invertebrates, mammals, plants, and microbes [14, 37, 38]. However, the commercially available ones are isolated from plant, fungal sources or derived from bacterial sources recombinantly [39–41].
Our laboratory has been elucidating the lysosomal enzymes and their sorting receptors in several species of invertebrates with a long-term goal of delineating evolutionarily conserved mechanisms of lysosomal biogenesis [5, 29, 34, 42–44]. Earlier reports on Hex from our group have shown that the freshwater mussel Unio, Lamellidens marginalis as well as Lamellidens corrianus possesses both HexA and HexB, which are the major forms of Hex isoenzymes characterized in mammals including humans. Further, our studies in a freshwater cnidarian Hydra - which is known for its extraordinary regenerative properties and as an excellent model to understand regeneration, tissue remodelling, and autophagy – have demonstrated its potential utility as a model organism towards understanding lysosomal biology [29, 30]. Our earlier report comparing lysosomal glycosidases such as Hex, acid phosphatase, and β-glucuronidase from three different strains of Hydra (viz., Hydra vulgaris Ind- Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1) was the first study on the biochemical characterization of these enzymes from Hydra [30].
Some of the initial experiments carried out to understand the biochemical properties of Hex using crude soluble extracts of H. vulgaris Ind- Pune revealed the pH optima (6.0), the temperature optima (60 ºC) and the KM value (1.3 mM). Furthermore, whole-mount in situ hybridization experiments indicated the localization of Hex to be on the endodermal region of the body column of Hydra suggesting its possible potential role in digestion. The biochemical properties and pattern of localization of Hex were similar in the other two strains of Hydra that were investigated [30]. This study provided a base for our present work, where we aimed to first purify and biochemically characterize Hex in greater detail from the H. vulgaris Ind- Pune strain.
The initial enzyme assays carried out with Hydra soluble extract in this study confirmed the presence of several lysosomal hydrolases, and the Hex activity was found to be significantly high, which is consistent with our earlier report [30]. Further, the immunoblotting experiments using soluble extract suggested that the isoform of Hex in H. vulgaris Ind- Pune is HexB. Unlike several other species studied including humans, where the presence of two to three isoforms of Hex (HexA, HexB, and HexS) are evident [8, 34], our results hint at HexB being the major isoform present in this strain of Hydra, though further extensive studies are essential to confirm the presence of other isoforms. Further, a two-step sequential chromatographic method involving phenyl-Sepharose hydrophobic interaction chromatography followed by S-200 gel filtration chromatography resulted in the purification of HexB to homogeneity. A very similar purification strategy with CM-Toyopearl 650M (hydrophobic interaction) chromatography followed by Toyopearl HW-55F (size exclusion) chromatography was employed for the purification of β-Hex from the moon jellyfish- Aurelia aurita, one of the very few cnidarian members from which Hex has been purified and characterized [45].
In a 10% SDS-PAGE, the purified Hydra HexB migrated as a single band of approximate molecular weight of 65 kDa, that was confirmed by immunoblotting (reactivity with human antibody) of the purified enzyme. This is very similar to the Hex purified from Aurelia aurita which had also showed a band of 65 kDa in SDS-PAGE. The native molecular weight of Aurelia aurita was estimated to be 130 kDa by size exclusion chromatography, indicating that the moon jellyfish Hex was a homodimer [45]. Interestingly, Hex from another marine cnidarian Zoanthid Palythoa caribaeorum showed an apparent molecular mass of 25 kDa [46]. The HexB purified from Unio (L. corrianus) had a molecular mass in the range of 55–60 kDa, whose native molecular mass was found to be ~ 130 kDa [34]. In a 10 % native PAE Hydra HexB migrated as a single band with a molecular mass of ~ 130 kDa. Since HexB is known to be an evolutionarily conserved homodimer of β subunits coded by the gene HEXB [37, 47], the Hydra HexB purified in the present study is likely to be also a homodimer. This was indeed confirmed by homology modelling using the amino acid sequence of Hydra HexB and comparing it with the crystal structure of human HexB as the template. The predicted model for Hydra HexB showed the homodimeric nature of Hydra HexB as expected. This suggests topological conservation of the domain architecture of the enzyme till higher vertebrates including humans. Moreover, the authenticity of the purified Hydra HexB was also confirmed by zymogram analysis using specific fluorogenic substrate 4-methylumbelliferyl-β-D-glucosaminide.
The purified Hydra HexB showed wobbling specificity with respect to substrates as the enzyme cleaved both pNPβGlcNAc and pNPβGalNAc, a characteristic feature of hexosaminidases [36, 48]. At optimum temperature, HexB showed relatively higher activity with pNPβGlcNAc, whereas at optimum pH the activity was relatively high with pNPβGalNAc. Depending on the source of the enzyme, the activity ratio is known to vary [48] as in the case of Hex from Palythoa caribaeorum, which showed most activity towards pNPGlcNAc and only about 40% activity with pNPGalNAc [46].
The optimum pH for Hydra HexB was found to be in the range of 5.0 to 6.0 which agrees with the acidic milieu of the lysosomal lumen, where all the glucosyl hydrolases function. The pH optima for Hex from other cnidarians such as A. aurita and P. caribaeorum were observed to be 4.0 and 5.0, respectively [45, 46]. While HexB from Unio showed maximum activity at pH 4.0, Hex from the soil bacterium Stackebrandtia nassauensis exhibited peak activity at pH 6.0 [34, 49]. Hex purified from Sf-9 cell cultures of the lepidopteran insect Spodoptera frugiperda showed maximum activity at pH 5.5 [14].
Furthermore, Hydra HexB showed a narrow range of pH stability as it was stable only at pH 5.0 and the activity significantly declined upon incubation in more acidic or alkaline ranges, with inactivation of enzyme at pH below 3.0 and above 8.0. This is very similar to the pH stability exhibited by jellyfish Hex [45]. On the other hand, HexB from L. corrianus displayed a broad range of pH stability (3.0–7.0) [34]. Similarly, remarkable pH stability was also observed in Hex purified from prawn, Penaeus vannamei, where the enzyme was stable in a broad pH range of 4.2 to 10.0, with maximum activity at pH 5.2 [16].
Temperature optima for HexB purified in this study was in the range of 50 ºC to 60 ºC, which is similar to Hex from A. aurita (50 ºC) and P. caribaeorum (45 ºC to 60 ºC) [45, 46]. While the optimum temperature for HexB purified from Unio, L. marginalis was in the range of 60 ºC to 70 ºC, HexB from L. corrianus had a specific temperature optimum of 60 ºC [34, 42] The Hex purified from prawn, however, showed maximum activity at 45 ºC [16].
Interestingly, the thermal stability studies in Hydra HexB indicated that the enzyme was stable only up to 40 ºC followed by a rapid decline in activity. The thermal stability studies of Hex characterized from several other species including A. aurita, P. vannamei, and L. corrianus, indicate that the stability of the enzyme is up to 40 ºC, corroborating our observation [16, 34, 45]. While, SnHex from the bacterium S. nassauensis was thermally stable up to 50 ºC [49], HexB from L. marginalis exhibited unusual thermal stability (up to 80 ºC) [42].
Several studies characterizing Hex from various sources have studied the effects of metal ions on enzyme activity. Interestingly, the addition of divalent cations had no effect on the activity of Hydra HexB (data not shown) which is remarkably similar to what was observed in SnHex from the bacterium S. nassauensis [49].
While our study provides valuable insights into the biochemical properties of the purified Hydra HexB, further investigation aiming at extensive biophysical characterization, protein sequencing, functional studies, and post-translational modifications would be essential to gain further insights into Hydra HexB. Additionally, exploring the possible presence of other Hex isoforms could broaden our understanding of lysosomal enzyme diversity in Hydra.