Comparative analysis of β-hexosaminidase and acid phosphatase from Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1: Localization studies of acid phosphatase and β-hexosaminidase from H. vulgaris Ind-Pune

Highlights

• Differential expression of lysosomal enzymes in three Hydra strains

• Enzyme analysis revealed high levels of β-hexosaminidase and acid phosphatase.

• Hexosaminidase showed maximum activity at an acidic pH.

• Differential affinity of β-hexosaminidase and acid phosphatase in Hydra

• Predominant endodermal expression of lysosomal enzymes

Abstract

The present report describes a comprehensive study on comparative biochemical characterization of two lysosomal enzymes, acid phosphatase and β-hexosaminidase in three different strains of Hydra; Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1 (self-feeder-1). Since morphology and habitat of Hydra effect lysosomal enzymes and their response to environmental pollutants, it would be interesting to identify them in different Hydra strains so as to use them as toxicity testing. Preliminary studies revealed a differential expression of acid phosphatase, β-hexosaminidase and β-glucuronidase in three Hydra strains. Expression of all three lysosomal enzymes in H. vulgaris Ind-Pune was low in comparison to H. vulgaris Naukuchiatal and H. magnipapillata sf-1, while their expression is comparable in H. vulgaris Naukuchiatal and H. magnipapillata sf-1. The Michaelis-Menten (K_M) values for lysosomal β-hexosaminidase using 4-nitrophenyl N-acetyl-β-D-glucosaminide as substrate were found to be 1.3 mM, 1.1 mM and 0.8 mM, respectively for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1. For acid phosphatase using 4-nitrophenyl-phosphate as substrate, the K_M values were 0.38 mM, 1.2 mM and 0.52 mM respectively, for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and sf-1 strains. The optimum temperature for β-hexosaminidase was 60 °C for H. vulgaris Ind-Pune, while 50 °C was observed for H. vulgaris Naukuchiatal and sf-1 strains. The optimum pH for β-hexosaminidase was found to be 6.0 for H. vulgaris Ind-Pune and H. vulgaris Naukuchiatal, and 5.0 for sf-1. The optimum temperature and pH of acid phosphatase was similar in all three strains, viz., 40 °C and 3.0, respectively. Preliminary localization studies using whole mount in situ hybridization revealed predominant endodermal expression of three enzymes in H. vulgaris Ind-Pune. Our results thus support the conservation of lysosomal hydrolases in Hydra.

Introduction

The degradative functions of the cell are carried out by specialized organelles called lysosomes. Lysosomal hydrolases degrade extra cellular molecules; pathogens internalized by endocytosis or phagocytosis, and also aid in the turnover of intracellular proteins and maintain the cellular homeostasis and differentiation (Stoka et al., 2016; de Marcos Lousa and Denecke, 2016). Further, lysosomal enzymes also show important roles in tissue remodeling, membrane repair, cell adhesion, immune function, pigmentation, and signaling. Specially, lysosomal acid phosphatases, β-glucuronidase, acid sphingomyelinase, and proteases such as cathepsins, participate in tissue repair, dedifferentiation, aging, immune response, and other processes.

Acid phosphatases in the vertebrates are a distinct family of enzymes that cause hydrolysis of phosphomonoesters at an acidic pH of 3.0–5.0. Different types of acid phosphatases have been identified till date, based on their structure, catalytic and immunological properties, cellular localization and tissue distribution (Suter et al., 2001). Lysosomal acid phosphatase (EC 3.1.3.2, LAP), a ubiquitously expressed enzyme is often considered as a key biochemical marker enzyme for lysosomes (De Duve, 1983). Synthesized as a membrane-bound precursor with 7–8 N-linked oligosaccharides, a transmembrane domain and a cytoplasmic tail, it recycles from the early endosomes to the plasma membrane (Braun et al., 1989). Once the precursor enzyme reaches lysosomes, it is proteolytically processed in the lysosomal matrix and a mature LAP is released (Gottschalk et al., 1989). Though LAP is majorly localized to lysosomes in different organs, other forms of soluble acid phosphatase, specifically expressed and secreted in the prostate gland have also been identified. Both LAP and prostate acid phosphatases are glycoproteins and contain mannose and glucosamine in the carbohydrate moiety, show identical subunits of molecular weight of 48–52 kDa, and are sensitive to L-tartrate inhibition (Lemansky et al., 1985). Subsequently, a tartrate resistant type-5 acid phosphatase (Acp5), an orthophosphoric monoesterase has also been identified in lysosomal compartments of mononuclear phagocytes and osteoclasts (Hayman et al., 2000; Bevilacqua et al., 1991). Absence of either of these acid phosphatases, LAP/Acp5 leads to mild phenotypes, suggesting partial compensatory mechanisms by the other phosphatase. However, deficiency of both phosphatases (LAP/Acp5) leads to abnormal lysosomal storage in soft and mineralized tissues.

Another equally important lysosomal enzyme includes β-N-acetylhexosaminidase (EC 3.2.1.52, Hex), which catalyzes the hydrolysis of terminal N-acetylhexosamine residues from the non-reducing ends of glycoconjugates (Venugopal and Sivakumar, 2013). Three isozymes of β-N-acetylhexosaminidase composed of two subunits, α and β are commonly identified: Hex A (α-β), Hex B (β-β), and Hex S (α-α). Hex A and Hex B are functionally more significant, while Hex S is a minor form and shows less activity (Hepbildikler et al., 2002). Both Hex A and Hex B are synthesized as precursors and are transported to lysosomes and depend on the recognition of mannose residues by mannose phosphate receptors. Deficiency of Hex A leads to Tay-Sachs disease, while deficiency of both isozymes, Hex A and B causes Sandhoff disease. In addition to these three isozymes, a relatively less known β-hexosaminidase, HexD, encoded by HexDC gene also exists (Gutternigg et al., 2009). However, the function of this nuceocytoplasmic localized enzyme is not clearly understood (Alteen et al., 2016).

Several reports have demonstrated the role of LAP and Hex during regeneration. Coward and his group (Coward et al., 1973) have demonstrated an increase in LAP activity during planarian regeneration. LAP activity was also found to be high during tail regeneration in lizards (Alibardi, 1998) and in tail regression during metamorphosis in Xenopus tadpoles (Robinson, 1970, Robinson, 1972). Role of LAP in dedifferentiation, a condition prerequisite for limb regeneration was also detected in the adult urodele (Miller and Wolfe, 1968). Biochemical and immunohistochemical investigations also have demonstrated a prominent increase of LAP activity during retinoic acid mediated limb regeneration and dedifferentiation in regenerating salamander larvae (Ju and Kim, 1994, 2004). Similarly, role of hexosaminidase during regeneration has been well demonstrated. Role in chitin degradation by hydrolyzing glycosidic bonds of 2-acetamido 2-deoxy β-D-glycosides, along with chitinases in invertebrates has been identified (Cohen, 2009). Role of N-acetyl β-D-hexosaminidase (N-acetylglucusoaminidase and N-acetylgalactosaminidase) during dedifferentiation in the first 24 h of regenerating planarians has also been demonstrated (Pascolini et al., 1981). These results point towards the involvement of lysosomal hydrolases in regeneration, tissue remodeling and differentiation.

Our laboratory mainly focuses on delineation of lysosomal biogenesis pathway in vertebrates and invertebrates. Previous reports from our laboratory have established the evolutionary conservation of lysosomal enzymes as well as their sorting receptors from molluscs to vertebrates (Kumar and Bhamidimarri, 2015). Recently we have also identified lysosomal hydrolases and mannose-6 phosphate receptor dependent lysosomal targeting system for the first time in a simple diploblastic Cnidarian, ‘Hydra’ (Bhamidimarri et al., 2018).

In order to understand the evolutionary conservation of lysosomal enzymes and their biogenesis pathway, particularly in the Cnidarians, it is important to carry out a systematic study on the enzymes and their biochemical nature. These studies would eventually allow us to establish the conservation of lysosomal biogenesis in the animal kingdom. Hydra, a fresh water Cnidarian, has been used as a powerful model system in biology to dissect molecular mechanisms underlying different processes such as wound healing, regeneration, immune response and autophagy. Presence of unique features, such as, remarkable power of regeneration, absence of cellular senescence and maintenance of axial polarity, has resulted in using Hydra as a suitable model to study morphogenesis and pattern formation. Since lysosomal enzymes play crucial roles in various physiological and stressed conditions, Hydra can act as a powerful experimental model to study their involvement during tissue remodeling and regeneration. Further, the availability of RNA seq analysis (Wenger et al., 2019) and identification of molecular map and differentiation trajectories of specific lineages would help in elucidating developmental mechanisms at single-cell level in response to environmental and experimental perturbations (Siebert et al., 2019). The ready availability of different strains of ‘Hydra’ in our laboratory also has prompted us to look more closely into the details of various lysosomal enzyme activities in each strain. Since each of them was obtained from three different habitats, it is expected that these strains express different levels of enzymes and may respond differently to environmental pollutants. Hence it is interesting to identify the differences, if any, in the lysosomal enzyme activities in these strains and may help in using them as potential toxicological indicators.

In the present paper we characterized two lysosomal hydrolases, β-N-acetylhexosaminidase and acid phosphatase biochemically and carried out comparative analysis among three different strains of Hydra, Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1. H. vulgaris Ind-Pune and H. vulgaris Naukuchiatal are distinct Indian strains belonging to the ‘vulgaris’ group of hydra. Though, both strains belong to same ‘vulgaris’ species, they show significant morphological and taxonomical variations (Londhe et al., 2017). H. magnipapillata sf-1 is a temperature sensitive mutant strain of Japanese H. magnipapillata that grows normally at 18 °C, while at restrictive temperatures, at 28 °C or more, the polyps loses interstitial stem cells when maintained for 8–10 days (Sugiyama and Fujisawa, 1978). In this paper, we report comparative analysis of β-N-acetylhexosaminidase and acid phosphatase in three Hydra strains, which showed significant differences in their biochemical properties. This suggests possible differences in their cellular and tissue makeup and may thus point towards their differences in physiological adaptations to the external environment.