Purification of 57kDa Hyaluronidase from the venom of Conus betulinus (Linnaeus, 1758)

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Purification of 57kDa Hyaluronidase from the venom of Conus betulinus (Linnaeus, 1758)

https://doi.org/10.21203/rs.3.rs-2004455/v1

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The enzyme hyaluronidase cleaves the substrate hyaluronic acid. In the study, hyaluronidase was isolated from the venom gland of C. betulinus and characterised using SDS-PAGE, FTIR, and HPLC. The protein content of crude venom is approximately 4mg/ml, whereas purification with Sepacryl S-100 yielded 0.04mg/ml protein with 0.463TRU/mg specific activity. The detected hyaluronidase had a molecular weight of 57kDa when compared to a standard protein marker. The presence of a peak at Rt 57.23 as hyaluronidase is revealed by HPLC analysis, and the wavelength pattern is similar to the standard bovine testicular hyaluronidase.

Hyaluronidase (HAase) is an endoglycosidase that hydrolyses the glycosaminoglycan hyaluronic acid (HA) with a repeating unit of D-glucuronic acid and N-acetyl glucosamine that is not bound to the proteoglycan. The HA polymers are ejected onto the glycocalyx or into the extracellular framework (ECM). HA was integrated by HA synthases, which polymerize HA on the surface of the intracellular layer. HA exists fundamentally in skin, primarily in mammalian dermis, and is corrupted by some distinct HAase in well-ordered physical cells (Harunari et al., 2014).

Many small predators use a chasing strategy that relies on exceptionally strong and fast acting venoms to incapacitate and kill their prey. Hyals have been identified in venoms ranging from vertebrates such as snakes, stonefish, and reptiles to spineless creatures such as honey bees, scorpions, and creepy crawlies. When all is said and done, the expected capacity of venom Hyals is the degradation of ECM polysaccharides in the attacked creature's connective tissue, inevitably resulting in misfortune of basic uprightness (Sainio, 2020). As a result, venom Hyals are frequently depicted as spreading factors that encourage the appropriation of other venom parts through tissues, which is critical for quick immobilisation of prey as well as resistance against various predators. Several studies have looked into the spreading action of venom Hyals on the ECM of vertebrates (Biner et al, 2015). In vivo dermonecrosis probes rabbit skin with recombinant creepy crawly venom Hyal (Loxo scales intermedia) in combination with recombinant dermonecrotic poison LiRecDT1 clearly demonstrates this catalyst's capacity as a "spreading factor" (Valéria et al, 2013).

HAase is found in a variety of organisms, including mammals, bacteria (Streptomyces (Ohya et al. 1970), Streptococcus (Hamai et al. 1989)), bacteriophage (Baker et al. 2002), and venom from terrestrial bees (Kameny et al. 1984), Hornets (Kolarich et al. 2005), scorpions Pes The only acid-active accelerator in the class circulation is HAase. Eukaryotic HAase is classified into three types: neutral-active endo—N-glucosaminidase, acidic-active endo—N-glucosaminidase, and endo—glucuronidase. Anti-inflammatory and anti-allergic drugs target HAase at the molecular level. Furthermore, anti-allergic agents such as disodium cromoglycate and tranilast, as well as anti-inflammatory agents such as glycyrrhizin, have HAase inhibitory activity (Sakamoto et al. 1980). Furthermore, the histamine-releasing agent 48/80 activates HAase (Kakegawa et al. 1985). As a result, HAase inhibitors have the potential to be used as an anti-inflammation drug. The current study seeks to isolate hyaluronidase from the marine snail C.betulinus.

2.1. Venom Ducts Collection:

The C. betulinus specimens were transferred aseptically into a sterile room for dissection. The shell was broken with a hammer, and the venom ducts were extracted. The venom ducts were immediately processed for further research.

2.2. Crude Extract Isolation:

The crude extract was extracted from their venom duct and homogenised with sodium phosphate buffer (PH 7). The fresh venom ducts were homogenised with 2 mL of buffer in an ice bath using a motor and pestle. The mixture was centrifuged at 4°C for 10 minutes at 5000 rpm. The supernatant (a crude extract) was collected and stored at -20°C for further investigation.

2.3 Protein Estimation

The protein content of crude venom was determined using the Lowry et al. (1951) method and BSA as a standard.

2.4. Hyaluronidase Purification from C. betulinus venom:

The crude venom (5 ml) was loaded onto a Sephacryl S-100 column (2.5 x 48 cm) that had previously been pre-equilibrated with 50 mM sodium acetate buffer, pH 6.0, containing 100 mM NaCl, and eluted with the 1.5 M NaCl buffer at a flow rate of 40 ml/h. At 280 nm, fractions of 2.0 ml were collected and monitored. The highest hyaluronidase activity fractions were pooled and dialyzed.

2.5 RP-HPLC Detection of Cono-Hyal:

Partially purified venom fractions with the highest hyaluronidase content from a single specimen of C. betulinus were pooled and adjusted to a final volume of 20L in 0.1% trifluoroacetic acid before being separated by RP-HPLC on a 4.6 mm x 250 mm, 4.6-m-particle diameter, 100-pore size C18 column (Phenomenex, Torrance, CA, USA) with a 0.1% TFA (Solvent A) and 0.1% TFA in acetonitrile were used as buffers (Solvent B). For 60 minutes, the venom was separated using an incremental linear gradient of 1% B/min. The chromatogram was recorded at 236nm using a PDA detector and the standard Hyaluronidase at the same time.

2.6 Hyaluronidase Assay:

The turbidometric method of Poh et al. was used to determine hyaluronidase enzyme activity (1992). The test solution was incubated with hyaluronic acid (0.5 mg/mL in 0.05 M acetate buffer, pH 4.5, containing 0.15 M NaCl) for 15 minutes at 37°C (final volume 0.5 mL), and the reaction was stopped by adding 1 mL of 2.5% (w/v) cetyltrimethylammonium bromide in 2% (w/v) NaOH. At 400 nm, the absorbance of each reaction mixture was measured. The activity to reduce turbidity was expressed as a percentage of the remaining hyaluronic acid. The absorbance was set to 100% because no enzyme was added. The amount of enzyme required to hydrolyze 50% of 50 g hyaluronic acid is expressed as turbidity reducing units. One unit is defined as the amount of enzyme required to reduce turbidity by the same amount as 1.0 U of international standard preparation. One volume of acetate buffer (0.05 M acetate buffer, pH 4.5, containing 0.15 M NaCl) and two volumes of stop solution were used to make the blank.

2.7. Determination of Molecular Weight:

The molecular weight of isolated Hyaluronidase was determined using a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) system and the Laemmli method (1970).

2.8 Assay of Hyaluronidase Plate:

Richman and Baer's previously described plate assay was used (1980). A 10 mL solution of 3% (w/v) agarose in 0.05Mm sodium acetate buffer (pH 5.3) was melted in a boiling water bath and cooled to 60o C before being added to a 10 mL solution of 2 mg/mL HA and chondroitin sulphate (CS) in a 60o C bath with constant stirring. The solution was quickly poured into a petri dish after being stirred for about 1 minute to ensure complete mixing. Suction was used to empty cylindrical holes 2 mm in diameter punched into the gel. In the punched well, 10 l of hyaluronidase was poured. The plates were incubated at 34o C for 20 hours. The buffer alone served as the control. After incubating for 30 minutes, the plate was covered with 10 mL of 10% (w/v) cetylpyridinium chloride in water. Following that, the circle diameters were measured with a calliper against a dark background.

2.9. FTIR Characterization:

For the presence of functional groups in the sample, Fourier transform infrared spectroscopy (FTIR) spectra were obtained. All spectra were obtained using an infrared spectrophotometer (Shimadzu) with a data acquisition rate of 2 cm-1 per point from 4000 to 400 cm-1.

3.1. Protein Calculation:

The crude venom's protein concentration was estimated to be 4 mg/ml.

3.2. Hyaluronidase enzyme column purification:

The crude venom was applied to a gel filtration column made of Sephacryl S-100. While the sample was eluted with 1.5 M NaCl, 2ml of each fraction was eluted and the absorbance at 280nm was measured. The fractions (F3 to F10) in this result show the presence of protein content. Despite the fact that these fractions were estimated for hyaluronidase activity, the collected fractions (F14-F19) exhibit strong hyaluronidase activity (Fig. 1.)

3.3 RP-HPLC detection of Cono-Hyal from C. betulinus

Figure 2 depicts the RP-HPLC analysis of the injected fraction of C. betulinus. Both standard hyaluronidase and C. betulinus detected the peak at 57.70 min.

3.4. Activity and yield of hyaluronidase:

Using hyaluronidase as a standard, the hyaluronidase activity of C. betulinus was estimated to be 0.954 and 0.462 TRU (given that 0. 25l degraded 50% of HA). Table 1

3.5. Plate assay for hyaluronidase:

To demonstrate hyaluronidase activity, a HA and CS substrate-agarose plate assay was performed (Fig. 3). The hyaluronidase activity was identified as a zone formed by assimilation of the substrate exhibit on the agarose gel. The activity that causes the zone to expand corresponds to the amount of hyaluronidase in the agarose gel.

3.6 SDS page:

The electrophoretic profile of hyaluronidase stained with silver staining was shown (Figure. 4). The 12% gel was used to estimate the molecular weight of crude venom and purified hyaluronidase from C. betulinus, which was found to be approximately 57 kDa when compared to standard protein molecular weight markers (range 205 to 29 kDa).

3.7 FTIR analysis

FTIR analysis revealed the major functional groups of Standard hyaluronidase and C. betulinus hyaluronidase (Fig. 5A-5B and Table. 2). The major Peaks of standard and purified hyaluronidase have the same wave number range. The presence of functional groups in standard and C. betulinus hyaluronidase is shown in Table 2.

The Hyaluronidase enzyme was isolated from a variety of sources, and its presence in human tissues, animal venoms, and other pathogenic organisms has been documented. The acrosome membrane of sperm, which plays a critical role in mammalian fertilisation, is structurally similar to that of animal venom Hyaluronidase. Thus, venom hyaluronidase is assigned the scattering function by facilitating the spread of toxins in connective tissue via glucosamine glycan hydrolysis, resulting in systemic poisoning (Sutti et al., 2014). Hyaluronidase applications in biomedical fields are expected to grow year after year; the use of venom hyaluronidases will broaden the range of applications in emerging modern medicine.

The crude venom was extracted using phosphate buffer saline in the current study, and the protein content was found to be 4mg/ml. Similarly, Giji et al., 2014 determined the protein content of C. betulinus in lyophilized powder to be 0.9mg/ml. The variation in the protein content of the sample was caused by the extraction method. Saravanan et al. (2009) calculated the protein content of C. figulinus crude toxin extract to be 1.9 mg/ml. This indicates that the protein yield was sufficient for further purification of the enzyme and its activity.

Hyaluronidase was purified from crude venom in two steps using gel filtration column chromatography and ethanol precipitation methods. The activity-based fractions were pooled together after column purification. The first step was Sephacryl S-100 gel filtration chromatography, which resulted in two variations of peaks for protein content and Hyaluronidase activity in conus venom gland extract. Using the same Sephacryl S-100 gel filtration chromatography, 0.65g of hyaluronidase was extracted from sting ray venom (Magalhaes et al., 2008). The purification of hyaluronidases from Stonefish Synanceja horrida yielded similar results (Poh et al., 1995).

The yield and activity of Hyaluronidase enzyme from C.betulinus were 0.04mg and 0.462TRU, respectively. Because of the lower content of targeted enzyme in Conus venom, the enzyme yield was considered to be the minimum yield after purification. Hyaluronidase cleaves HA at random, and the products of each degradation reaction are suitable substrates for further digestion. As a result, hyaluronidase generates a diverse set of HA fragments. Low molecular weight HA fragments have the potential to be involved in inflammation, immune stimulation, and angiogenesis. Furthermore, venom hyaluronidases cause tissue damage and inflammation in connective tissues and blood vessels (Feng et al., 2008).

Following that, it was discovered that the hyaluronidases family, including HYAL1 and HYAL2, have broad substrate specificity and depolymerize not only HA but also chondroitin sulphate and dermatan sulphate (Yamamoto et al., 20017). As a result, the C. betulinus Hyaluronidase fraction exhibited significant enzymatic activity in degrading HA and CS, with a specific activity discovered to cleave the tetrasaccharides and hexasaccharides. As a result, the current finding indicates that conus Hyaluronidase's GAG depolymerizing activity is specific for both HA and CS. Similarly, the same method of degrading HA by Hyaluronidase was discovered in C. consors (Violette et al., 2012) and C. purpurascens (Mller et al., 2017). This distinguishing feature was so distinct and notable that the dispersing factor of Hyaluronidase has previously been used in the therapeutic area to improve the efficacy of some other drugs and treatment approaches in clinical trials (Violette et al., 2012).

In any medium, such as paper or starch gel, the polyacrylamide gel electrophoresis method is more convenient. Protein electrophoresis in polyacrylamide gel (PAGE) was performed in both buffer and Sodium dodycle Sulphate (denaturing). Separation in buffer gel is dependent on both the charge and the size of the protein, whereas separation in SDS gel is solely dependent on the size. Protein analysis and comparison in a large number of samples is simple on polyacrylamide gel slabs. SDS PAGE analysis revealed that the 57 kDa protein in C. betulinus has a single band and exists as a monomer. Similarly, a 79 kDa hyaluronidase was isolated from Potamotrygon motoro stingray venom. Furthermore, the 62 kDa hyaluronidases were discovered in the Stonefish Synanceja horrida (Poh et al., 1992). As a result, other conus species, such as C. consors and C. purpurascens, show 60 KDa and 50 kDa molecular ranges of enzyme in 2D gel electrophoresis (Violette et al., 2012; Moller et al., 2017). The majority of hyaluronidase is a monomer that varies depending on the species.

When compared to a standard from bovine testicular, the FT-IR spectrum of hyaluronidase revealed conformational-sensitive bands. The presence of OH and -NH stretching vibrations was represented by a peak with a wave number of 3336.85 cm-1. The presence of CH2 asymmetric and symmetric stretching is implied by the 2360.87 cm-1 band. Furthermore, the same peak range was discovered in standard Hyaluronidase. The amide-1 bands of the Hyaluronidase at 1690 − 1520 cm1 are primarily C-O stretching vibrations with some N-H bending and C-H stretching vibrations, the amide I bands at 1700 − 1610 cm1 are primarily C = O stretching vibrations with some N-H bending and C-H stretching vibrations, and the amide II bands at 1575 − 1480 cm1 are primarily N-H bending vibrations with some CN stretching vibrations. The 1515 cm1 band, which corresponds to tyrosine residues, was easily identified, whereas the bands at 1585 and 1565 cm1 could correspond to aspartate and glutamate carboxy residues, respectively (Silvia et al., 2006).

The disulphide bond in the enzyme of Conus species was denoted by the wave numbers that exist at 474.49 cm1. Disulphide bonds have a protein-independent stabilising role and are important for enzyme activity. While the presence of disulphide bonds generally improves protein stability, they can also counteract the local instability effects of hydrophobic forces in many cases (Evison et al., 2009). The interaction of the tertiary structure, disulphide bonds, and hydrophobic core residues allows hyaluronidases to finish their biological process.

It is concluded that the current study is a step toward developing a potential enzyme from a venomous marine organism with novel therapeutic applications. The isolated C. betulinus conus hyaluronidase enzyme will be scaled up with pharmaceutical industries for commercial hyaluronidase production in the future.

Acknowledgments and funding statement: The authors would like to thank the Department of Science and Technology (DST), Government of India, for financial support under the programme 'National Facility for Marine Natural Products and Drug Discovery Research' [G4 (2) /21343], as well as the Annamalai University administration for providing necessary facilities.

Conflicts of interest: The authors declared that there is no conflicts of interest.

Human/animal study statement: This study involves no animal/humans as part of the research.

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TABLE.1 The yield of Hyaluronidase

Purification
Weight
Protein/ activity

Crude venom duct
1.5g
4mg/ml

Sephacryl S-100
0.04mg
0.462 TRU/mg

Tables 2 is not available with this version

No competing interests reported.

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Purification of 57kDa Hyaluronidase from the venom of Conus betulinus (Linnaeus, 1758)

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Processes

2.1. Venom Ducts Collection:

2.2. Crude Extract Isolation:

2.3 Protein Estimation

2.4. Hyaluronidase Purification from C. betulinus venom:

2.5 RP-HPLC Detection of Cono-Hyal:

2.6 Hyaluronidase Assay:

2.7. Determination of Molecular Weight:

2.8 Assay of Hyaluronidase Plate:

2.9. FTIR Characterization:

3. Results

3.1. Protein Calculation:

3.2. Hyaluronidase enzyme column purification:

3.3 RP-HPLC detection of Cono-Hyal from C. betulinus

3.4. Activity and yield of hyaluronidase:

3.5. Plate assay for hyaluronidase:

3.6 SDS page:

3.7 FTIR analysis

4. Discussion

5. Conclusion

Declarations

References

Tables

Additional Declarations

Status:

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Purification	Weight	Protein/ activity
Crude venom duct	1.5g	4mg/ml
Sephacryl S-100	0.04mg	0.462 TRU/mg