Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology

Abstract

Hyaluronidases are enzymes that mainly degrade hyaluronan, the major glycosaminoglycan of the interstitial matrix. They are involved in several pathological and physiological activities including fertilization, wound healing, embryogenesis, angiogenesis, diffusion of toxins and drugs, metastasis, pneumonia, sepsis, bacteremia, meningitis, inflammation and allergy, among others. Hyaluronidases are widely distributed in nature and the enzymes from mammalian spermatozoa, lysosomes and animal venoms belong to the subclass EC 3.2.1.35. To date, only five three-dimensional structures for arthropod venom hyaluronidases (Apis mellifera and Vespula vulgaris) were determined. Additionally, there are four molecular models for hyaluronidases fromMesobuthus martensii, Polybia paulista and Tityus serrulatus venoms. These enzymes are employed as adjuvants to increase the absorption and dispersion of other drugs and have been used in various off-label clinical conditions to reduce tissue edema. Moreover, a PEGylated form of a recombinant human hyaluronidase is currently under clinical trials for the treatment of metastatic pancreatic cancer. This review focuses on the arthropod venom hyaluronidases and provides an overview of their biochemical properties, role in the envenoming, structure/activity relationship, and potential medical and biotechnological applications.

Hyaluronidase; Scorpion; Spider; Caterpillar; Hymenoptera; Insects; Cloning; Heterologous expression; PEGylation; Biotechnological applications

Introduction

Hyaluronidases are glycosidases that cleave preferentially the hyaluronan in the extracellular matrix (ECM) found in soft connective tissues. Hyaluronan is a linear polysaccharide formed by repeating disaccharide units of N-acetyl-β-D-glucosamine (GlcNAc) and β-D-glucuronic acid (GlcUA) linked via alternating β-1,3 and β-1,4 glycosidic bonds (Fig. 1). It acts as an impact absorber and lubricant in the articulations, playing a relevant structural role in maintaining the architecture of the ECM. This is rendered possible since hyaluronan interacts with many water molecules, assuming great viscoelasticity [¹]–[³].

Fig. 1
Structure of hyaluronan. The repeating disaccharide units of N-acetyl-β-D-glucosamine (GlcNAc) and β-D-glucuronic acid (GlcUA) linked via alternating β-1,3 (highlighted in green) and β-1,4 glycosidic bonds (highlighted in red) are shown. The hyaluronidases EC 3.2.1.36 cleave the β-1,3 glycosidic bond, EC 3.2.1.35 the β-1,4 glycosidic bond and the EC 4.2.2.1 the β-1,4 glycosidic bond by elimination, yielding a double bond between carbons 4’ and 5’

Hyaluronidases increase up to 20 times the infusion rates and penetration of molecules up to 200 nm in diameter because of the cleavage of hyaluronan, reducing the obstacle that the interstitial matrix presents to fluid and drug transfer [⁴].

The hyaluronidase activity was identified for the first time by Duran-Reynals in 1928, but the term hyaluronidase was introduced only in 1940 [⁵], [⁶]. These enzymes are widely distributed in nature and have been reported in animal venoms (such as snake [⁷], [⁸], wasp [⁹], scorpion [¹⁰],[¹¹], bee [¹²], hornet [¹³], freshwater stingray [¹⁴], fish [¹⁵], spider [¹⁶], lizard [¹⁷] and caterpillar [¹⁸], [¹⁹] venoms), human organs (testis, eye, skin, spleen, liver, kidneys, uterus) and corporal fluids (placenta, tears, blood, sperm) [²⁰], [²¹], bacteria [²²], hookworm [²³], fungi[²⁴], bacteriophages [²⁵], crustaceans [²⁶], mollusks [²⁷], leeches [²⁸], other animal tissues[²⁹], [³⁰] and malignant tumors [³¹]. The first hyaluronidase was isolated from bovine testis [²⁹] and has been legally sold in the USA since 1948 [³²], [³³]. However, the first venom hyaluronidase was isolated only in 1973 from Dugesiella hentzi tarantula venom [³⁴]. Usually, hyaluronidases are present in venoms in such low proportion that they are not detectable through proteomic analyses [³⁵].

Hyaluronidases are classified into three major groups [²¹], [³⁶], [³⁷]. They degrade preferentially hyaluronan, though different reaction mechanisms are involved (Fig. 2). The first group (EC 3.2.1.35) includes vertebrate enzymes (e. g. mammalian and venom hyaluronidases) that are endo-β-N-acetyl-D-hexosaminidases and hydrolyze the β-1,4 glycosidic bond between GlcNAc and GlcUA residues in hyaluronan to the tetrasaccharide (GlcUA-GlcNAc-GlcUA-GlcNAc) as the main product. These enzymes are also able to cleave chondroitin sulfate. The second group (EC 3.2.1.36) is composed of hyaluronidases from annelids, such as leeches and certain crustaceans. These enzymes are endo-β-D-glucuronidases that degrade hyaluronan to the tetrasaccharide (GlcNAc-GlcUA-GlcNAc-GlcUA) by hydrolyzing the β-1,3 glycosidic bond between GlcUA and GlcNAc residues in hyaluronan. The third one (EC 4.2.2.1, former EC 4.2.99.1) is represented by bacterialN-acetyl-D-hexosaminidases that cleave the β-1,4 glycosidic bond by a beta elimination reaction, degrading hyaluronan, chondroitin sulfate and dermatan sulfate to disaccharides with a double bond between carbons 4 and 5.

Fig. 2
The three major groups of hyaluronidases. The EC numbers, catalysis, substrates, main products and sources of each hyaluronidase group are shown

The hyaluronidase activity is modulated by various activators (adrenalin, histamine and acid phosphatase found in prostate, liver, kidney, erythrocytes and platelets) and inhibitors (antihistamines, salicylates, heparin, dicoumarin, vitamin C and flavonoids) [³⁸], [³⁹].

This enzyme has been used as an adjuvant to increase the absorption and dispersion of injected drugs [³²], [⁴⁰], to reduce edema [⁴¹], [⁴²] and local side effects in tissues [³²], and as a healing-promoting agent for skin lesions [⁴³]. In 2005, a highly purified recombinant human hyaluronidase (rHuPH20) was approved by the FDA [³²], [⁴⁴]. A phase IV clinical trial using this enzyme associated to insulin analogs is under study for the treatment of type 1 diabetes [⁴⁵],[⁴⁶]. Additionally, a biopharmaceutical product containing rHuPH20 was approved for the treatment of adult patients with primary immunodeficiency in 2014 [⁴⁰], and another one containing a PEGylated form of rHuPH20 (PEGPH20) has been under a phase II clinical trial for the first-line treatment of metastatic pancreatic cancer [⁴⁷].

Many hyaluronidases (from prokaryotes and eukaryotes) have been studied and a great diversity in their activity can be observed among different species. Such diversity has been demonstrated by the optimal pH, isoelectric point, number of isoforms, molecular mass, substrate specificity and sensitivity in the presence of various modulators [⁴⁸].

Hyaluronidases are usually classified as acid-active (maximum activity from pH 3 to pH 4) or neutral-active enzymes (maximum activity from pH 5 to pH 6) [⁴⁹]. Hyaluronidases isolated from snake, bee and scorpion venoms are active in pH from 4 to 6 and present a molecular mass between 33 and 100 kDa [⁵⁰]–[⁵²]. Cevallos et al. [⁵⁰] observed that venom hyaluronidases from some invertebrates (Dolichovespula maculata, Vespula germanica, Pogonomyrmex rugosus andCentruroides limpidus limpidus) presented less than 50 kDa, while those from vertebrates (bovine,Heloderma horridum horridum, H. suspectum suspectum, Lachesis muta, Crotalus basiliscus,Bothrops asper and Micrurus nigrocinctus) are comprised of hyaluronidases larger than 60 kDa and more than one active isoform may be present. On the other hand, small hyaluronidases (lower than 60 kDa) have already been identified in vertebrate venoms [⁷] and enzymes presenting more than 50 kDa have already been isolated from invertebrate ones [⁵³].

About two-thirds of all named species in the world, which corresponds to approximately 1,000,000 species, belong to the phylum Arthropoda and the class Insecta represents about 80 % of this phylum. The arthropods have significant economic impact and affect all aspects of the human life. Examples include the pollination of crops and diseases spread by insects and ticks [⁵⁴]. The present paper reviews the hyaluronidases present in arthropod venoms as well as their potential applications in medicine and biotechnology.

Review

Role of arthropod venom hyaluronidases in envenoming

Hyaluronidases are not toxic by themselves, but they potentiate the effect of other toxins present in venoms, contributing to the local and systemic effects of envenoming [¹⁶], [⁵⁵]. Furthermore, they are described as allergens from arthropod venoms, being able to induce severe and fatal anaphylactic IgE-mediated reactions in humans [¹³], [⁵⁶]. These enzymes are known as “spreading factors”, a concept firstly introduced by ^{Duran-Reynals in 1933} [¹¹], [⁵⁷]. This action was experimentally confirmed [¹⁷], resulting in the hydrolysis of hyaluronan and chondroitin sulfates A and C, which promotes the diffusion of toxins through the tissues and blood circulation of the victim/prey [⁷], [⁸], [¹⁷], [⁵⁸], [⁵⁹].

The hyaluronidase plays a key role in the Pararama associated phalangeal periarthritis observed after the envenoming caused by the caterpillar Premolis semirufa[⁶⁰]. The enzyme from the spider Hippasa partita indirectly potentiated the myotoxicity of VRV-PL-VIII myotoxin and the effect of hemorrhagic complex-I [¹⁶]. Similar results were observed with the recombinant hyaluronidase from the spider Loxosceles intermedia, which increased the effect of the recombinant dermonecrotic toxin LiRecDT1 [⁵⁵]. The enzyme from telmophage insects is responsible for extending the feeding lesion and diffusing anti-hemostatic agents into the host tissue [⁶¹].

Additionally, the hyaluronidase from Tityus serrulatus scorpion venom potentiates the activity of Ts1, the major neurotoxin present in this venom, increasing the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) [¹⁰]. Therefore, to assess the importance of hyaluronidase in the scorpion envenoming process, the toxic effects of T. serrultatus venom were evaluated after the in vitro and in vivo inhibition and immunoneutralization of the hyaluronidase activity by anti-hyaluronidase serum produced in rabbits [⁶²]. In vivoneutralization assays using anti-hyaluronidase serum inhibited or delayed death of mice. The use of aristolochic acid, a pharmacological inhibitor of hyaluronidase, also inhibited death. On the other hand, the survival of mice was reversed after the addition of native hyaluronidase to pre-neutralized venom, showing that hyaluronidase plays a critical role in systemic envenoming [⁶²]. Therefore, inhibitors of the hyaluronidase activity are potential first aid agents to treat envenoming cases [⁶²], [⁶³].

Structure of hyaluronidases

There are 128 and 92 known primary sequences deposited in the NCBI and UniProt databanks, respectively, for hyaluronidases belonging to 53 genera divided into the classes Arachnida, Chilopoda and Insecta from the phylum Arthropoda (Table 1). All deposited sequences were evidenced at transcript level, with the exception of those from Phoneutria, Tityus andDolichovespula, which were evidenced at protein level.

Table 1
Hyaluronidases from the phylum Arthropoda

Class	Order	Members	Family	Genus	Number of entries (NCBI; UniProt)	Ref.
Arachnida	Araneae	Spiders	Ctenidae	Phoneutria	1; 1 - F a	—
			Sicariidae	Loxosceles	2; 1	[55]
			Theraphosidae	Brachypelma	1; 1 - F	[82]
	Scorpiones	Scorpions	Bothriuridae	Cercophonius	1; 1 - F	[129]
			Buthidae	Hottentotta	1; 1 - F	[130]
				Isometroides	1; 1 - F	[129]
				Mesobuthus	3; 1	[76]
				Tityus	5; 5 - F*(2)	[62], [74],[131]
			Urodacidae	Urodacus	1; 1 - F	[129]
Chilopoda	Geophilomorpha	Centipedes	Linotaeniidae	Strigamia	0; 2	—
Insecta	Blattodea	Termites	Rhinotermitidae	Coptotermes	1; 1	—
	Coleoptera	Beetles	Curculionidae	Dendroctonus	2; 2 - F	[132]
			Tenebrionidae	Tribolium	2; 1	[133]
	Diptera	Biting horseflies	Tabanidae	Tabanus	1; 1	[134]
		Biting midges	Ceratopogonidae	Culicoides	4; 4	[135]–[138]
		Black flies	Simuliidae	Simulium	1; 1	[139]
		Midges	Chaoboridae	Corethrella	1; 1	[140]
		Mosquitos	Culicidae	Aedes	6; 4	[141]
				Anopheles	4; 2	[142], [143]
				Culex	9; 5	—
				Psorophora	1; 1 - F	[144]
		Moth flies	Psychodidae	Lutzomyia	2; 2	—
				Phlebotomus	4; 4	[145]–[147]
	Hemiptera	Aphids	Aphididae	Acyrthosiphon	1; 1	—
		Assassin bug	Reduviidae	Rhodnius	0; 3 - F	—
				Triatoma	1; 1 - F	—
	Hymenoptera	Ants	Formicidae	Acromyrmex	1; 1	[148]
				Atta	0; 1	[149]
				Camponotus	1; 1	[150]
				Cerapachys	1; 1	[151]
				Harpegnathos	1; 1	[150]
				Solenopsis	1; 1 - F	[152]
		Bees	Apidae	Apis	13; 3	[64], [118]
				Bombus	2; 0	—
			Megachilidae	Megachile	1; 0	—
		Parasitoid wasps	Braconidae	Chelonus	4; 4 - F	—
				Glyptapanteles	5; 5	—
				Meteorus	1; 1	—
				Microplitis	3; 3	—
		Pteromalid parasitoid wasps	Pteromalidae	Nasonia	1; 0	—
		Spider wasps	Pompilidae	Anoplius	1; 1	—
		Wasps	Vespidae	Dolichovespula	3; 1 - a	[13]
				Eumenes	1; 1	[153]
				Orancistrocerus	1; 1	[154]
				Polistes	2; 1 - F	—
				Polybia	2; 2 - F	[9]
				Rhynchium	1; 1	—
				Vespa	2; 1	[155]
				Vespula	12; 6	[65]
	Isoptera	Dampwood termites	Termopsidae	Zootermopsis	1; 1 - F	[156]
	Lepidoptera	Butterflies	Nymphalidae	Danaus	2; 2	[157]
		Silkmoths	Bombycidae	Bombyx	1; 1	[158]
	Phthiraptera	Lice	Pediculidae	Pediculus	8; 4	[159]
TOTAL					128; 92

• ^aEvidence at protein level (all the others at transcript level); F: fragment; —: unpublished

The first three-dimensional (3D) structure reported for a hyaluronidase belonging to the family 56 of glycoside hydrolases was reported for the enzyme from Apis mellifera venom in 2000 [PDB: 1FCQ; 1FCU; 1FCV] [⁶⁴]. The overall topology of hyaluronidases from this family resembles a classical (β/α) _n triosephosphate isomerase (TIM) barrel, where n is equal to 8 in the hyaluronidase from A. mellifera venom and 7 in those from Vespula vulgaris [PDB: 2ATM] and P. paulista [Pp–Hyal, PMDB: PM0077230] venoms [⁹], [⁶⁴], [⁶⁵].

Snake and human hyaluronidases present five disulfide bonds [⁸], [⁶⁶]. The disulfide bonds Cys332–Cys343, Cys336–Cys371 and Cys373–Cys383 are part of the epidermal growth factor-like (EGF-like) domain [⁶²]. The enzymes from A. mellifera, V. vulgaris and P. paulista venoms show two disulfide bonds (Cys17–Cys307 and Cys183–Cys196) [⁹], [⁶⁴], [⁶⁵], which are located in the catalytic domain and well conserved in venom hyaluronidases [⁶²]. On the other hand, the enzymes from T. serrulatus venom (TsHyal-1 and TsHyal-2, whose numbers of deposit were not stated) exhibit six disulfide bonds common to all known Arachnida hyaluronidases [⁶²]. The sixth disulfide bond (Cys172–Cys215), found only in the Arachnida hyaluronidases, may reinforce the stability of their catalytic site [⁶²].

On the basis of N-glycosylation, the recombinant hyaluronidase from L. intermedia presents four putative N-glycosylation sites in its structure; the enzyme from A. mellifera venom shows one of four possible sites [⁵⁵], [⁶⁴]. The one from V. vulgaris venom has three of five possible sites, the one from P. paulista venom shows three putative glycosylation sites, the BmHYI from Mesobuthus martensii venom presents five potential N-glycosylation sites (the number of deposit for the molecular model was not stated), while TsHyal-1 and TsHyal-2 from T. serrulatus venom has seven and ten putative glycosylation sites, respectively [⁹], [⁶²], [⁶⁵], [⁶⁷].

Besides the fact that N-glycosylation sites are not conserved between TsHyal-1 and TsHyal-2, the isoforms from T. serrulatus venom show a variation in the active site groove in position 219. TsHyal-1 has a tyrosine (Y), while TsHyal-2 has a histidine (H) at the same position, which may cause different substrate specificity [⁶²]. A mutation in the positioning residue Y247 in human Hyal-4 (equivalent to Y219 in TsHyal-1) altered the substrate specificity [⁶⁸]. Among the known primary sequences of hyaluronidase, only TsHyal-2 has a histidine (H) in the position 219 [⁶²].

The residues Ser299, Asp107, and Glu109, located at surface-exposed regions of the Pp-Hyal (P. paulista hyaluronidase) structure, on opposite sides of the cavity, interact with the polar hydroxyl nitrogen atoms of hyaluronan and with potential antibody-binding sites (five conformational and seven linear epitopes located at surface-exposed regions of the structure) [⁹]. These residues are of great importance for substrate transport into the active site through electrostatic interactions with the carboxylic groups of hyaluronan. Three amino acid residues (Asp107, Phe108, Glu109, according to the Pp-Hyal sequence) are extremely conserved and present in the active sites of all hyaluronidases [⁹]. Only the 3D-structure from A. mellifera hyaluronidase (Api m 2) was solved with the substrate hyaluronan, enabling the identification of the active site and points of contact with the substrate [⁹]. In Api m 2, the residues Asp111 and Glu113 are highly conserved in the substrate-binding site and are proton donors essential for the catalysis [⁶⁴]. The structure of the complex enzyme-substrate suggests an acid–base catalytic mechanism, in which Glu113 is the proton donor and the N-acetyl group of hyaluronan acts as the nucleophile [⁶⁴].

The residues Asp111, Tyr184, Trp301 are essential for the positioning of the substrate’s carbonyl of the acetamido group [²¹]. Tyr227 is responsible for the specificity for hyaluronan and Cys227 substitution is responsible for the chondroitinase function [²¹].

Arachnida venom hyaluronidases

Scorpion venom hyaluronidases

Scorpion venom hyaluronidases were first identified in 1975 in the venom of the South Indian scorpion Heterometrus scaber[⁶⁹]. Although several studies have demonstrated the presence of hyaluronidases in scorpion venoms, few studies have reported their isolation from these sources[⁷⁰]–[⁷²]. This may happen because hyaluronidases are difficult to isolate, only small amounts of them are found in venoms (when compared to other toxins) and their enzymatic activity is abolished very easily [⁷³]. These enzymes were isolated for the first time in 1990 from the venom of H. fulvipes in two chromatographic steps: molecular exclusion and cation-exchange chromatography [⁵³]. Six hyaluronidases were isolated from H. fulvipes[⁵³], T. serrulatus[¹⁰],[⁶²], Palamneus gravimanus[¹¹], T. stigmurus[⁷⁴] and M. martensii[⁷⁵] venoms and had their biochemical and structural characterization performed.

Currently, the application of “omics” techniques has enabled the identification of new compounds present in animal venoms. There are 12 and 10 known primary sequences deposited in the NCBI and UniProt databanks, respectively, for scorpion hyaluronidases (Table 1). Only two of them correspond to complete sequences: one from T. serrulatus venom [Swiss-Prot: W0HFN9] and the other from M. martensii venom [Swiss-Prot: P86100] [⁶²], [⁷⁶]. These protein sequences were deduced from cDNA sequences.

The molecular mass of scorpion venom hyaluronidases may range from 45 to 82 kDa [¹⁰], [⁵³],[⁶²]. Generally, they show maximum activity in pH between 4 and 6 and temperatures from 30 to 37 °C. Considerable loss of the hyaluronidase activity is observed at temperatures above 40 °C[¹⁰], [¹¹], [⁵³], [⁷⁵]. The hyaluronidase activity can also be inhibited by heparin, as reported for the enzyme from the scorpions H. fulvipes, P. gravimanus and M. martensii[¹¹], [⁵³], [⁷⁵]. Furthermore, dithiothreitol (DTT), some ions such as Cu ²⁺ and Fe ³⁺ , and flavonoids are also able to inhibit the hyaluronidase activity [¹⁰], [⁵³], [⁷⁵]. Interestingly, the activity of these enzymes may vary among different species and changes may occur in a diet-dependent manner [⁷⁷], [⁷⁸]. However, distinct geographical areas had no influence on the enzyme activity [⁷⁹].

Spider venom hyaluronidases

The first spider hyaluronidases, that are similar to the testicular enzyme, were reported in the venoms of the Brazilian species Lycosa raptoral and Phoneutria nigriventer in 1953 [⁸⁰]. However, the first spider venom hyaluronidase was only isolated in 1973 from the tarantula Dugesiella hentzi(Girard) and was reported as the major constituent of this venom [³⁴]. Other spider venom hyaluronidases were isolated from Loxosceles reclusa[⁸¹], Hippasa partita[¹⁶], Bracchypelma vagans[⁸²] and Vitaluis dubius[⁸³]. Additionally, the hyaluronidase activity was detected in several other spider venoms [⁸⁴]–[⁸⁹]. Moreover, three spider venom hyaluronidases from L. leata[⁹⁰],Bracchypelma vagans[⁸²] and L. intermedia[⁵⁵] were expressed in heterologous systems.

There are four and three known primary sequences deposited in the NCBI and UniProt databanks, respectively, for spider hyaluronidases (Table 1). The complete sequence of the enzyme from L. intermedia [Swiss-Prot: R4J7Z9] was obtained from its venom gland transcriptome [⁵⁵]. The enzyme from P. keyserlingi [Swiss-Prot: P86274] had the first 32 amino acid residues from its N-terminal identified by Edman degradation [⁹¹].

Spider venom hyaluronidases present a molecular mass that ranges from 33 to 47 kDa in their monomeric form [¹⁶], [³⁴], [⁵⁵], [⁸¹]–[⁸³] and maximum enzymatic activity at 37 °C in pH from 4 to 6 [¹⁶], [³⁴], [⁸³], [⁹²]. Spider venom hyaluronidases also show high specificity to hyaluronan, weak activity upon chondroitin sulfate A and an almost absence of activity upon chondroitin sulfates B and C [⁵⁵], [⁸²], [⁸³]. The activity of these hyaluronidases is inhibited by metal ions, such as Fe ³⁺ and Cu ²⁺ , divalent cations, temperatures above 60 °C and extreme levels of pH (under 4 and over 8) [¹⁶], [⁸¹], [⁸³]. The processes of thawing and freezing do not seem to influence the stability of the enzyme from D. hentzi and H. partita, whereas the enzyme from V. dubius venom had its activity decreased after a series of thawing and lyophilization cycles[¹⁶], [³⁴], [⁸³], [⁸⁹].

Chilopoda venom hyaluronidases

Centipedes contain a venom gland connected to a pair of forcipules which are used to capture preys. Centipede bites usually cause burning pain, paresthesia, edema and lead to superficial necrosis in human victims [⁹³]. The hyaluronidase activity has also been detected in the scolopendrid centipede venoms [⁹⁴]. The venoms from Otostigmus pradoi and Scolopendra viridicornis showed hyaluronidase-active bands of 40–66 kDa and an additional band of 32 kDa was detected in the first venom [⁹³], [⁹⁴]. There are two complete primary sequences deposited to theStrigamia genus in the Uniprot databank (Table 1) although no paper has been published yet.

Insecta venom hyaluronidases

Caterpillar venom hyaluronidases

The larvae of butterflies and moths are called caterpillars. They produce venom in order to protect themselves against predators that are envenomed upon touching them. The composition of the venom is not well known and it varies among different species of caterpillars [⁹⁵]. The presence of hyaluronidases has been reported in the venoms of Lonomia obliqua, Premolis semirufa andMegalopyge urens[¹⁸], [¹⁹], [⁶⁰]. The hyaluronidase activity of the P. semirufa venom was measured in the presence of hyaluronan [⁶⁰]. A hyaluronidase was suggested as the factor behind the Pararama associated phalangeal periarthritis, a serious public health problem among the Brazilian tappers (rubber plantation workers). It is a disease associated with joint immobilization, loss of the cartilage and bone structure and is known to be caused by the P. semirufa envenoming[⁶⁰].

Additionally, lonoglyases are two hyaluronidases found in the L. obliqua venom that present 49 and 53 kDa [¹⁹]. These enzymes are endo-β-N-acetyl-D-hexosaminidases able to degrade hyaluronan and chondroitin sulfate. Lonoglyases show optimal activity from pH 6 to 7 and no activity was detected below pH 5 and over pH 8. Gouveia et al. [¹⁹] suggest that the ability of cleaving hyaluronan and chondroitin sulfate linked to the extracellular matrix could explain the effects of the venom, changing the cell adhesion and migration events. Some researchers have speculated that the degradation of the extracellular matrix results from the synergistic effect with other L. obliqua venom toxins, leading to local hemorrhage and renal failure [¹⁹].

Diptera venom hyaluronidases

Hyaluronidase is related to the hematophagic habit of telmophage insects, being found in the saliva of species of the genera Phlebotomus and Lutzomyia (Table 1). This enzyme extends the feeding lesion and diffuses anti-hemostatic agents into the host tissue, resulting in a microhemorrhage caused by the bite and facilitating the acquisition of blood by the insect [⁶¹]. The salivary hyaluronidase may facilitate the spreading of vector-borne microorganisms transmitted by blackflies (Simuliidae), biting midges (Ceratopogonidae) and horse flies (Tabanidae) [⁶¹].

Hymenoptera venom hyaluronidases

Proteins from social Hymenoptera (bees, wasps, and ants) venoms can trigger serious allergenic reactions in humans, such as pain, itching, inflammation and irritation, which in some cases may lead to death [⁹⁶]. The hyaluronidase is among the best-studied components from the Apis genus[⁹⁶]. The apian hyaluronidase is a basic glycoprotein (pI 9.0) of 41 kDa rich in aspartic and glutamic acids, containing 7.24 % carbohydrate [¹²].

Pp-Hyal (P. paulista hyaluronidase) is a glycosyl hydrolase comprised of 338 amino acids and shares high sequence identity (80 to 90 %) with wasp venom hyaluronidases of the Northern hemisphere. The mature enzyme presents a theoretical pI of 8.77 and mass of 43,277 Da determined by mass spectrometry analysis [⁹]. Four isoforms of hyaluronidase were identified in the P. paulista venom by two-dimensional SDS-PAGE followed by mass spectrometry [⁹⁷]. A 3D structural model of the most abundant isoform (Hyal III) was constructed. This isoform contains 288 amino acid residues, 44,340 Da and pI of 9.5 [⁹⁸]. The comparison between the Hyal III and Pp-Hyal also showed differences in 27 amino acid residues, in the number of disulfide bonds and in the tertiary structure [⁹]. The levels of hyaluronidase activity in Hymenoptera venoms vary in response to physiological and environmental factors and the presence of isoforms may be an important strategy to mislead the immune system [⁹]. The absence of carbohydrate moieties in the bee recombinant hyaluronidase polypeptide chain did not change its antibody binding. On the other hand, this structural difference causes protein aggregation due to the partial destabilization of the molecule [⁹⁹]. A heterogeneous pattern of N-glycosylation of the hyaluronidase Ves v 2 from V. vulgaris was shown by mass spectrometry, disclosing peptides with three different patterns of glycosylation sites: one with glycosylation in the positions Asn79 and Asn127; another in the positions Asn79 and Asn99 and the third one with only one glycosylation site in the position Asn99. Because of this variation, the in vitro diagnosis of allergic individuals to wasp venom is quite complex [¹⁰⁰].

Heterologous arthropod venom hyaluronidases

Hyaluronidases from different organisms have been expressed in various expression systems such as bacteria, yeast, plants, insects and mammalian cells [²⁸], [⁵⁵], [⁶⁵], [⁸²], [¹⁰¹]–[¹⁰⁸].

The first recombinant hyaluronidase ever produced was the Dol m 2, one of the major allergens from the white face hornet Dolichovespula maculata[¹³]. The recombinant Dol m 2 compared to a native hyaluronidase from the bee venom showed a common T cell epitope, which may be one of the reasons why some patients have sensitivity after bee and hornet envenoming [¹³]. The bee venom enzyme is the most well-characterized hyaluronidase from venoms. It was expressed in 1998 by Soldatova et al. [¹⁰⁹] in insect cells, making possible the determination of the first venom hyaluronidase crystal and the characterization of N-glycans by mass spectrometry [⁶⁴], [¹¹⁰].

Potential medical and biotechnological applications of arthropod venom hyaluronidases

There are some reports on the medical applications and off-label use of hyaluronidase in several medical fields [³²], [⁴⁰]. Additionally, some hyaluronidases have been studied to enhance the therapeutic index and the local diffusion of anticancer drugs into tissues and tumors [³⁸], [⁷⁵],[¹¹¹]–[¹¹⁷]. Among the arthropod venom hyaluronidases, BmHYA1 (a hyaluronidase isolated fromButhus martensi scorpion venom) reduced the expression of CD44 variant 6 in the breast cancer cell line MDA-MB-231 [⁷⁵].

Furthermore, a hyaluronidase from bee venom was complexed with IgG antibody, which allows the hyaluronidase’s epitope to be recognizable by the antibody and may contribute to the development of novel proteins with reduced immunogenicity to be used as a safer allergen-specific immunotherapy [¹¹⁸]. Recombinant allergens have been used for diagnostic and therapeutic purposes since they are obtained with consistent quality and unlimited amount [¹¹⁹]. Besides that, they can be modified to reduce their allergenicity and to promote beneficial immunologic properties with the aim of reducing IgE-mediated side effects after immunotherapy [¹¹⁹]–[¹²¹]. Distinct allergens which are absent or underrepresented in therapeutic venom preparations may play a key role for the success of immunotherapy [¹²²]. The immunoglobulin E (IgE), present in the serum of allergic patients to the Polybia paulista wasp venom, can recognize the recombinant hyaluronidase from P. paulista (Pp-Hyal-rec) expressed in E. coli system [¹²³]. A heterologous glycosylated hyaluronidase, rVes v 2 from Vespula species, expressed in insect cells system, was used to identify wasp venom allergic patients. The specific diagnosis of allergic patients was improved using the basophil activation test (BAT) with the allergen rVes v 2 when compared to the respective specific IgE detection in vitro[¹²⁴]. Moreover, the carbohydrate epitopes present in the glycosylated insect cell-expressed Api m 2 are responsible for antigenic cross-reactivity to bee and wasp venoms [¹⁰⁴], [¹²⁵]. On the other hand, the nonglycosylated E. coli-expressed Api m 2 enabled the serologic discrimination of bee and wasp allergy, allowing the correct prescription of venom immunotherapy [¹²⁵]. These reports demonstrate that recombinant antigens, such as hyaluronidases, have a great immunogenic potential in allergy diagnosis and immunotherapy[¹²³]. In the future, molecules consisting of allergen-derived peptides bound to a viral carrier might be used for prophylactic and therapeutic allergy vaccination, since they are promising vaccines free of IgE- and T cell-mediated side effects [¹²⁶].

The intranasal administration of hyaluronidase (bovine or isolated from T. serrulatus venom) stopped bleomycin-induced lung injury and fibrosis, and decreased the TGF-β production and collagen deposition, which makes hyaluronidase a promising tool for the recruitment of autologous MSC-like cells to the lungs in the treatment of pulmonary fibrosis [¹²⁷]. This effect could be improved with the use of a delivery system of poly (D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) loaded with hyaluronidase (HYAL-MP) [¹²⁸].

Finally, inhibitors of the hyaluronidase activity may be used as potential first aid agents in antivenom therapies since the enzyme has a relevant role in systemic envenoming [⁶²].

Conclusions

Hyaluronidases are a frequent component from Arthropod venoms. They hydrolyze hyaluronan from the extracellular matrix, facilitating toxin diffusion into the tissues of the prey/victims. Although they are not toxins, they indirectly potentiate the toxicity of venoms. Arthropod venom hyaluronidases are potential adjuvants of anticancer drugs and promising tools for the recruitment of autologous MSC-like cells to the lungs in the treatment of pulmonary fibrosis and for the development of novel proteins to be used in allergy diagnosis and immunotherapy. The isolation and characterization of novel arthropod venom hyaluronidases can unravel much more about the role of these enzymes, which justifies the increasing interest on them and on the development of new hyaluronidase-containing drugs and biopharmaceutical products. Moreover, these studies can contribute to the development of more effective antivenom therapies.

Acknowledgements

This study received financial support from the State of São Paulo Research Foundation (FAPESP, grant n. 2011/23236-4; scholarship to GAW, n. 2014/06170-8; scholarship to FGA, n. 2011/12317-3), the National Council for Scientific and Technological Development (CNPq, 303689/2013-7) and the Support Nucleus for Research on Animal Toxins (NAP-TOXAN-USP, grant n. 12–125432.1.3).

References

• ¹
  El-Safory NS, Fazary AE, Lee CK. Hyaluronidases, a group of glycosidases: current and future perspectives Carbohydr Polym 2010; 81(2):165-81.
• ²
  Laurent TC. Biochemistry of hyaluronan Acta Otolaryngol Suppl 1987; 442:7-24.
• ³
  Laurent TC, Fraser JR. Hyaluronan FASEB J 1992; 6(7):2397-404.
• ⁴
  Bookbinder LH, Hofer A, Haller MF, Zepeda ML, Keller GA, Lim JE et al.. A recombinant human enzyme for enhanced interstitial transport of therapeutics J Control Release 2006; 114(2):230-41.
• ⁵
  Duran-Reynalds F. Exaltation de l’activité de virus vaccinal par les extraits de certains organs Compt Rend Soc Biol 1928; 9:6-7.
• ⁶
  Meyer K, Hobby GL, Chaffee E, Dawson MH. Relationship between “spreading factor” and hyaluronidase Proc Soc Exp Biol Med 1940; 44:294-6.
• ⁷
  Pukrittayakamee S, Warrell DA, Desakorn V, McMichael AJ, White NJ, Bunnag D. The hyaluronidase activities of some Southeast Asian snake venoms Toxicon 1988;26(7):629-37.
• ⁸
  Bordon KC, Perino MG, Giglio JR, Arantes EC. Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom Biochimie 2012; 94(12):2740-8.
• ⁹
  Justo Jacomini DL, Campos Pereira FD, Pinto JRAS, dos Santos LD, da Silva Neto AJ, Giratto DT et al.. Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis Toxicon 2013; 64:70-80.
• ¹⁰
  Pessini AC, Takao TT, Cavalheiro EC, Vichnewski W, Sampaio SV, Giglio JR et al.. A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids Toxicon 2001; 39(10):1495-504.
• ¹¹
  Morey SS, Kiran KM, Gadag JR. Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom Toxicon 2006; 47(2):188-95.
• ¹²
  Kemeny DM, Dalton N, Lawrence AJ, Pearce FL, Vernon CA. The purification and characterization of hyaluronidase from the venom of the honey bee, Apis melliferaEur J Biochem 1984; 139(2):217-23.
• ¹³
  Lu G, Kochoumian L, King TP. Sequence identity and antigenic cross-reactivity of white face hornet venom allergen, also a hyaluronidase, with other proteins J Biol Chem 1995; 270(9):4457-65.
• ¹⁴
  Magalhaes MR, da Silva NJ, Jr UCJ. A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: Isolation and characterization Toxicon 2008;51(6):1060-7.
• ¹⁵
  Poh CH, Yuen R, Chung MC, Khoo HE. Purification and partial characterization of hyaluronidase from stonefish (Synanceja horrida) venom Comp Biochem Physiol B 1992; 101(1–2):159-63.
• ¹⁶
  Nagaraju S, Devaraja S, Kemparaju K. Purification and properties of hyaluronidase from Hippasa partita (funnel web spider) venom gland extract Toxicon 2007;50(3):383-93.
• ¹⁷
  Tu AT, Hendon RR. Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor Comp Biochem Physiol B 1983; 76(2):377-83.
• ¹⁸
  Ardao MI, Perdomo CS, Pellaton MG. Venom of the Megalopyge urens (Berg) caterpillarNature 1966; 209(5028):1139-40.
• ¹⁹
  Gouveia AICB, da Silveira RB, Nader HB, Dietrich CP, Gremski W, Veiga SS. Identification and partial characterisation of hyaluronidases in Lonomia obliqua venom Toxicon 2005; 45(4):403-10.
• ²⁰
  Gold EW. Purification and properties of hyaluronidase from human liver. Differences from and similarities to the testicular enzyme Biochem J 1982; 205(1):69-74.
• ²¹
  Stern R, Jedrzejas MJ. Hyaluronidases: their genomics, structures, and mechanisms of action Chem Rev 2006; 106(3):818-39.
• ²²
  Hynes WL, Walton SL. Hyaluronidases of Gram-positive bacteria FEMS Microbiol Lett 2000; 183(2):201-7.
• ²³
  Hotez PJ, Narasimhan S, Haggerty J, Milstone L, Bhopale V, Schad GA et al.. Hyaluronidase from infective Ancylostoma hookworm larvae and its possible function as a virulence factor in tissue invasion and in cutaneous larva migrans Infect Immun 1992; 60(3):1018-23.
• ²⁴
  Shimizu MT, Jorge AO, Unterkircher CS, Fantinato V, Paula CR. Hyaluronidase and chondroitin sulphatase production by different species of Candida J Med Vet Mycol 1995; 33(1):27-31.
• ²⁵
  Hynes WL, Ferretti JJ. Sequence analysis and expression in Escherichia coli of the hyaluronidase gene of Streptococcus pyogenes bacteriophage H4489A Infect Immun 1989; 57(2):533-9.
• ²⁶
  Karlstam B, Ljunglöf A. Purification and partial characterization of a novel hyaluronic acid-degrading enzyme from Antarctic krill (Euphausia superba) Polar Biol 1991;11:501-7.
• ²⁷
  Violette A, Leonardi A, Piquemal D, Terrat Y, Biass D, Dutertre S et al.. Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of Conus venoms Mar Drgs 2012; 10(2):258-80.
• ²⁸
  Jin P, Kang Z, Zhang N, Du G, Chen J. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers Sci Rep 2014; 4:4471.
• ²⁹
  Freeman ME, Anderson P, Oberg M, Dorfman A. Preparation of purified hyaluronidase from bovine testis J Biol Chem 1949; 180(2):655-62.
• ³⁰
  Bollet AJ, Bonner WM, Nance JL. The presence of hyaluronidase in various mammalian tissues J Biol Chem 1963; 238:3522-7.
• ³¹
  Podyma KA, Yamagata S, Sakata K, Yamagata T. Difference of hyaluronidase produced by human tumor cell lines with hyaluronidase present in human serum as revealed by zymography Biochem Biophys Res Commun 1997; 241(2):446-52.
• ³²
  Dunn AL, Heavner JE, Racz G, Day M. Hyaluronidase: a review of approved formulations, indications and off-label use in chronic pain management Expert Opin Biol Ther 2010; 10(1):127-31.
• ³³
  Department of Health & Human Services: FDA Docket No. 2003P-0494/CP1. FDA 2004, http://www. fda.gov/ohrms/dockets/dockets/05p0134/05p-0134-cp00001-Tab-C-vol1.pdf webcite
  » http://www. fda.gov/ohrms/dockets/dockets/05p0134/05p-0134-cp00001-Tab-C-vol1.pdf
• ³⁴
  Schanbacher FL, Lee CK, Wilson IB, Howell DE, Odell GV. Purification and characterization of tarantula, Dugesiella hentzi (girard) venom Hyaluronidase Comp Biochem Physiol B 1973; 44(2):389-96.
• ³⁵
  Boldrini-Franca J, Correa-Netto C, Silva MM, Rodrigues RS, De La Torre P, Perez A et al..Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management J Proteomics 2010; 73(9):1758-76.
• ³⁶
  Meyer K, Rapport MM. Hyaluronidases Adv Enzymol Relat Subj Biochem 1952; 13:199-236.
• ³⁷
  Kreil G. Hyaluronidases - a group of neglected enzymes Protein Sci 1995; 4(9):1666-9.
• ³⁸
  Menzel EJ, Farr C. Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses Cancer Lett 1998; 131(1):3-11.
• ³⁹
  Li MW, Yudin AI, Van de Voort CA, Sabeur K, Primakoff P, Overstreet JW. Inhibition of monkey sperm hyaluronidase activity and heterologous cumulus penetration by flavonoids Biol Reprod 1997; 56(6):1383-9.
• ⁴⁰
  Biopharmaceutical products in the U.S. and European markets: U.S. Approvals, 2002-present. Biopharma. 2014. [http://www.biopharma.com/approvals.html]. Accessed December 1st, 2014.
  » http://www.biopharma.com/approvals.html
• ⁴¹
  Favorito LA, Balassiano CM, Costa WS, Sampaio FJB. Treatment of phimosis: Structural analysis of prepuce in patients submitted to topical treatment with betamethasone in association with hyaluronidase Eur Urol Suppl 2008; 7(3):704.
• ⁴²
  Johnsson C, Hallgren R, Elvin A, Gerdin B, Tufveson G. Hyaluronidase ameliorates rejection-induced edema Transpl Int 1999; 12(4):235-43.
• ⁴³
  Fronza M, Caetano GF, Leite MN, Bitencourt CS, Paula-Silva FW, Andrade TA et al..Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing PLoS ONE 2014; 9(11):e112297.
• ⁴⁴
  Yocum RC, Kennard D, Heiner LS. Assessment and implication of the allergic sensitivity to a single dose of recombinant human hyaluronidase injection: a double-blind, placebo-controlled clinical trial J Infus Nurs 2007; 30(5):293-9.
• ⁴⁵
  Morrow L, Muchmore DB, Hompesch M, Ludington EA, Vaughn DE. Comparative pharmacokinetics and insulin action for three rapid-acting insulin analogs injected subcutaneously with and without hyaluronidase Diabetes Care 2013; 36(2):273-5.
• ⁴⁶
  ClinicalTrials.gov. Consistent 1. Metabolic and safety outcomes of Hylenex recombinant (hyaluronidase human injection) preadministered at CSII infusion site in participants with type 1 diabetes mellitus (T1DM). http://clinicaltrials.gov/show/NCT01848990 webcite
  » http://clinicaltrials.gov/show/NCT01848990
• ⁴⁷
  ClinicalTrials.gov. PEGPH20 plus Nab-paclitaxel plus Gemcitabine compared with Nab-paclitaxel plus Gemcitabine in subjects with stage IV untreated pancreatic cancer (HALO-109-202). http://clinicaltrials.gov/show/NCT01839487 webcite
  » http://clinicaltrials.gov/show/NCT01839487
• ⁴⁸
  Mio K, Csóka AB, Nawy SS, Stern R. Detecting hyaluronidase and hyaluronidase inhibitors. Hyaluronan-substrate gel and -inverse substrate gel techniques Methods Mol Biol 2001; 171:391-7.
• ⁴⁹
  Girish KS, Shashidharamurthy R, Nagaraju S, Gowda TV, Kemparaju K. Isolation and characterization of hyaluronidase a “spreading factor” from Indian cobra (Naja naja) venom Biochimie 2004; 86(3):193-202.
• ⁵⁰
  Cevallos MA, Navarro-Duque C, Varela-Julia M, Alagon AC. Molecular mass determination and assay of venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Toxicon 1992; 30(8):925-30.
• ⁵¹
  Fiszer-Szafarz B. Hyaluronidase polymorphism detected by polyacrylamide gel electrophoresis. Application to hyaluronidases from bacteria, slime molds, bee and snake venoms, bovine testes, rat liver lysosomes, and human serum Anal Biochem 1984; 143(1):76-81.
• ⁵²
  Iwanaga S, Suzuki T. Enzymes in snake venom In: Snake venoms Lee CY, editor. Springer, Berlin; 1979: p.95-9.
• ⁵³
  Ramanaiah M, Parthasarathy PR, Venkaiah B. Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom Biochem Int 1990;20(2):301-10.
• ⁵⁴
  Mason KA, Losos JB, Singer SR, Raven PH, Johnson GB. Coelomate Invertebrates In:Biology 9th ed. Mason KA, Losos JB, Singer SR, Raven PH, Johnson GB, editors. McGraw-Hill, New York; 2011: p.666-92.
• ⁵⁵
  Ferrer VP, de Mari TL, Gremski LH, Silva DT, da Silveira RB, Gremski W et al.. A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich’s hyaluronidase): from cloning to functional characterization PLoS Negl Trop Dis 2013;7(5): Article ID e2206
• ⁵⁶
  Kolarich D, Léonard R, Hemmer W, Altmann F. The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgarisFEBS J 2005; 272(20):5182-90.
• ⁵⁷
  Duran-Reynals F. Studies on a certain spreading factor existing in bacteria and its significance for bacterial invasiveness J Exp Med 1933; 58(2):161-81.
• ⁵⁸
  Xu X, Wang XS, Xi XT, Liu J, Huang JT, Lu ZX. Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom Toxicon 1982;20(6):973-81.
• ⁵⁹
  Yingprasertchai S, Bunyasrisawat S, Ratanabanangkoon K. Hyaluronidase inhibitors (sodium cromoglycate and sodium auro-thiomalate) reduce the local tissue damage and prolong the survival time of mice injected with Naja kaouthia and Calloselasma rhodostoma venoms Toxicon 2003; 42(6):635-46.
• ⁶⁰
  Villas-Boas IM, Gonçalves-de-Andrade RM, Squaiella-Baptistão CC, Sant’Anna OA, Tambourgi DV. Characterization of phenotypes of immune cells and cytokines associated with chronic exposure to Premolis semirufa caterpillar bristles extractPLoS ONE 2013; 8(9): Article ID e71938
• ⁶¹
  Volfova V, Hostomska J, Cerny M, Votypka J, Volf P. Hyaluronidase of bloodsucking insects and its enhancing effect on Leishmania infection in mice PLoS Negl Trop Dis 2008; 2(9): Article ID e294
• ⁶²
  Horta CCR, Magalhaes BF, Oliveira-Mendes BBR. do Carmo AO, Duarte CG, Felicori LF, et al. Molecular, immunological, and biological characterization of Tityus serrulatus venom hyaluronidase: New insights into its role in envenomation. PLoS Negl Trop Dis 2014; 8(2): Article ID e2693
• ⁶³
  Girish KS, Kemparaju K. Inhibition of Naja naja venom hyaluronidase by plant-derived bioactive components and polysaccharides Biochemistry (Mosc) 2005; 70(8):948-52.
• ⁶⁴
  Markovic-Housley Z, Miglierini G, Soldatova L, Rizkallah PJ, Muller U, Schirmer T. Crystal structure of hyaluronidase, a major allergen of bee venom Structure 2000;8(10):1025-35.
• ⁶⁵
  Skov LK, Seppala U, Coen JJ, Crickmore N, King TP, Monsalve R et al.. Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom Acta Crystallogr D Biol Crystallogr 2006;62(Pt6):595-604.
• ⁶⁶
  Chao KL, Muthukumar L, Herzberg O. Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesisBiochemistry 2007; 46(23):6911-20.
• ⁶⁷
  Xia X, Liu R, Li Y, Xue S, Liu Q, Jiang X et al.. Cloning and molecular characterization of scorpion Buthus martensi venom hyaluronidases: a novel full-length and diversiform noncoding isoforms Gene 2014; 547(2):338-45.
• ⁶⁸
  Jedrzejas MJ, Stern R. Structures of vertebrate hyaluronidases and their unique enzymatic mechanism of hydrolysis Proteins 2005; 61(2):227-38.
• ⁶⁹
  Nair RB, Kurup PA. Investigations on the venom of the South Indian scorpion Heterometrus scaber Biochim Biophys Acta 1975; 381(1):165-74.
• ⁷⁰
  Wright RP, Chan TK, Honetschlager L, Howell DE, Odell GV. Enzymes and toxins of the scorpion venom Palamneus gravimanus Toxicon 1977; 15(3):197-205.
• ⁷¹
  Akhunov A, Chernetskaia II, Sadykov AS. Biochemical characteristics of the venoms of arthropods of Central Asia Dokl Akad Nauk SSSR 1985; 285(4):1009-11.
• ⁷²
  Basu A, Gomes A, Dasgupta SC, Lahiri SC. Histamine, 5-HT & hyaluronidase in the venom of the scorpion Lychas laevifrons (Pock) Indian J Med Res 1990; 92:371-3.
• ⁷³
  Stern R, Csoka AB. Mammalian hyaluronidases. Glycoforum/Science of Hyaluronan Today. 2000. http://www.glycoforum.gr.jp/science/hyaluronan/hapdf/HA15.pdf webcite
  » http://www.glycoforum.gr.jp/science/hyaluronan/hapdf/HA15.pdf
• ⁷⁴
  Batista CV, Roman-Gonzalez SA, Salas-Castillo SP, Zamudio FZ, Gomez-Lagunas F, Possani LD. Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species Comp Biochem Physiol C Toxicol Pharmacol 2007; 146(1–2):147-57.
• ⁷⁵
  Feng L, Gao R, Gopalakrishnakone P. Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensi Comp Biochem Physiol C Toxicol Pharmacol 2008; 148(3):250-7.
• ⁷⁶
  Feng L, Gao R, Meng J, Gopalakrishnakone P. Cloning and molecular characterization of BmHYA1, a novel hyaluronidase from the venom of Chinese red scorpion Buthus martensi Karsch Toxicon 2010; 56(3):474-9.
• ⁷⁷
  Venancio EJ, Portaro FC, Kuniyoshi AK, Carvalho DC, Pidde-Queiroz G, Tambourgi DV.Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms Toxicon 2013; 69:180-90.
• ⁷⁸
  Pucca MB, Amorim FG, Cerni FA, Bordon KC, Cardoso IA, Anjolette FA et al.. Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity Toxicon 2014; 90:326-36.
• ⁷⁹
  Rodríguez-Ravelo R, Coronas FI, Zamudio FZ, González-Morales L, López GE, Urquiola AR et al.. The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas J Venom Anim Toxins incl Trop Dis 2013; 19(1):13.
• ⁸⁰
  Kaiser E. Trypsin and hyaluronidase inhibitor of human serum; the inhibition of the proteolytic and hyaluronic acid cleavage enzymes of snake and spider venoms by human serum Biochem Z 1953; 324(5):344-50.
• ⁸¹
  Wright RP, Elgert KD, Campbell BJ, Barrett JT. Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider Arch Biochem Biophys 1973;159(1):415-26.
• ⁸²
  Clement H, Olvera A, Rodriguez M, Zamudio F, Palomares LA, Possani LD et al..Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom Toxicon 2012; 60(7):1223-7.
• ⁸³
  Sutti R, Tamascia ML, Hyslop S, Rocha-e-Silva TA. Purification and characterization of a hyaluronidase from venom of the spider Vitalius dubius (Araneae, Theraphosidae) J Venom Anim Toxins incl Trop Dis 2014; 20(1):2.
• ⁸⁴
  Savel-Niemann A. Tarantula (Eurypelma californicum) venom, a multicomponent system Biol Chem Hoppe Seyler 1989; 370(5):485-98.
• ⁸⁵
  Nagaraju S, Mahadeswaraswamy YH, Girish KS, Kemparaju K. Venom from spiders of the genus Hippasa: biochemical and pharmacological studies Comp Biochem Physiol C Toxicol Pharmacol 2006; 144(1):1-9.
• ⁸⁶
  Young AR, Pincus SJ. Comparison of enzymatic activity from three species of necrotising arachnids in Australia: Loxosceles rufescens, Badumna insignis and Lampona cylindrata Toxicon 2001; 39(2–3):391-400.
• ⁸⁷
  Barbaro KC, Knysak I, Martins R, Hogan C, Winkel K. Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas Toxicon 2005;45(4):489-99.
• ⁸⁸
  García-Arredondo A, Rodríguez-Rios L, Díaz-Peña LF, Vega-Ángeles R. Pharmacological characterization of venoms from three theraphosid spiders: Poecilotheria regalis Ceratogyrus darlingi and Brachypelma epicureanum J Venom Anim Toxins incl Trop Dis 2015; 21:15.
• ⁸⁹
  Rocha-e-Silva TA, Sutti R, Hyslop S. Milking and partial characterization of venom from the Brazilian spider Vitalius dubius (Theraphosidae) Toxicon 2009; 53(1):153-61.
• ⁹⁰
  Fernandes-Pedrosa MF, Junqueira-de-Azevedo IL, Gonçalves-de-Andrade RM, Kobashi LS, Almeida DD, Ho PL et al.. Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags BMC Genomics 2008; 9:279.
• ⁹¹
  Richardson M, Borges MH, Cordeiro MN, Pimenta AMC, de Lima ME, Rates B. Hyaluronidase from venom of Brazilian scorpion Tityus serrulatus. UniProtKB. 2008;P85841.
• ⁹²
  da Silveira RB, Chaim OM, Mangili OC, Gremski W, Dietrich CP, Nader HB et al..Hyaluronidases in Loxosceles intermedia (brown spider) venom are endo-beta-N-acetyl-D-hexosaminidases hydrolases Toxicon 2007; 49(6):758-68.
• ⁹³
  Malta MB, Lira MS, Soares SL, Rocha GC, Knysak I, Martins R et al.. Toxic activities of Brazilian centipede venoms Toxicon 2008; 52(2):255-63.
• ⁹⁴
  Undheim EA, King GF. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals Toxicon 2011; 57(4):512-24.
• ⁹⁵
  Kuspis DA, Rawlins JE, Krenzelok EP. Human exposures to stinging caterpillar: Lophocampa caryae exposures Am J Emerg Med 2001; 19(5):396-8.
• ⁹⁶
  De Lima PR, Brochetto-Braga MR. Hymenoptera venom review focusing on Apis mellifera J Venom Anim Toxins incl Trop Dis 2003; 9(2):149-62.
• ⁹⁷
  dos Santos LD, Santos KS, Pinto JR, Dias NB, de Souza BM, dos Santos MF et al.. Profiling the proteome of the venom from the social wasp Polybia paulista: A clue to understand the envenoming mechanism J Proteome Res 2010; 9(8):3867-77.
• ⁹⁸
  Pinto JR, Santos LD, Arcuri HA, Dias NB, Palma MS. Proteomic characterization of the hyaluronidase (E.C. 3.2.1.35) from the venom of the social wasp Polybia paulistaProtein Pept Lett 2012; 19(6):625-35.
• ⁹⁹
  Soldatova LN, Marcovic-Housley Z, Luong TN, Mueller UR, Slater J. The role of glycosylation in the IgE binding of the major honeybee allergen, hyaluronidase J Allergy Clin Immunol 2002; 109(1):S134-S.
• ¹⁰⁰
  Seppala UR, Mutenda K, Monsalve R, Skov L, King TP, Ipsen H et al.. Analysis of N-Glycosylation in Vespula vulgaris hyaluronidase Ves v 2 J Allergy Clin Immunol 2006;117(2):S119-S.
• ¹⁰¹
  Berry AM, Lock RA, Thomas SM, Rajan DP, Hansman D, Paton JC. Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli Infect Immun 1994; 62(3):1101-8.
• ¹⁰²
  Gmachl M, Kreil G. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm Proc Natl Acad Sci U S A 1993; 90(8):3569-73.
• ¹⁰³
  Bakke M, Kamei J, Obata A. Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp FEBS Lett 2011; 585(1):115-20.
• ¹⁰⁴
  King TP, Lu G, Gonzalez M, Qian N, Soldatova L. Yellow jacket venom allergens, hyaluronidase and phospholipase: Sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergyJ Allergy Clin Immunol 1996; 98(3):588-600.
• ¹⁰⁵
  Reitinger S, Boroviak T, Laschober GT, Fehrer C, Müllegger J, Lindner H et al.. High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris Protein Expr Purif 2008; 57(2):226-33.
• ¹⁰⁶
  Li H, Yang J, Chen Y, Guan L, Du L, Guo Y et al.. Expression of a functional recombinant oleosin-human hyaluronidase hPH-20 fusion in Arabidopsis thaliana Protein Expr Purif 2014; 103:23-7.
• ¹⁰⁷
  Jung Y, Jung MY, Park JH, Jung GC, Hong YS, Yeom CH et al.. Production of human hyaluronidase in a plant-derived protein expression system: plant-based transient production of active human hyaluronidase Protein Expr Purif 2010; 74(2):181-8.
• ¹⁰⁸
  Frost GI, Csoka AB, Wong T, Stern R. Purification, cloning, and expression of human plasma hyaluronidase Biochem Biophys Res Commun 1997; 236(1):10-5.
• ¹⁰⁹
  Soldatova LN, Crameri R, Gmachl M, Kemeny DM, Schmidt M, Weber M et al.. Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli J Allergy Clin Immunol 1998; 101(5):691-8.
• ¹¹⁰
  Soldatova LN, Tsai C, Dobrovolskaia E, Marković-Housley Z, Slater JE. Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells Allergy Asthma Proc 2007; 28(2):210-5.
• ¹¹¹
  Lee JH, Moore LD, Kumar S, Pritchard DG, Ponnazhagan S, Deivanayagam C.Bacteriophage hyaluronidase effectively inhibits growth, migration and invasion by disrupting hyaluronan-mediated Erk1/2 activation and RhoA expression in human breast carcinoma cells Cancer Lett 2010; 298(2):238-49.
• ¹¹²
  Civalleri D, Esposito M, De Cian F, Balletto N, Vannozzi MO, Mondini G et al.. Effects of adjuvant hyaluronidase in tumors refractory to chemotherapy. Review of the literature and pharmacokinetics of cisplatin after regional administration in animals and humans G Chir 1997; 18(4):175-81.
• ¹¹³
  Klocker J, Sabitzer H, Raunik W, Wieser S, Schumer J. Hyaluronidase as additive to induction chemotherapy in advanced squamous cell carcinoma of the head and neckCancer Lett 1998; 131(1):113-5.
• ¹¹⁴
  Baumgartner G, Gomar-Hoss C, Sakr L, Ulsperger E, Wogritsch C. The impact of extracellular matrix on the chemoresistance of solid tumors - experimental and clinical results of hyaluronidase as additive to cytostatic chemotherapy Cancer Lett 1998; 131(1):85-99.
• ¹¹⁵
  Spruss T, Bernhardt G, Schonenberger H, Schiess W. Hyaluronidase significantly enhances the efficacy of regional vinblastine chemotherapy of malignant melanomaJ Cancer Res Clin Oncol 1995; 121(4):193-202.
• ¹¹⁶
  Shuster S, Frost GI, Csoka AB, Formby B, Stern R. Hyaluronidase reduces human breast cancer xenografts in SCID mice Int J Cancer 2002; 102(2):192-7.
• ¹¹⁷
  Jacobson A, Rahmanian M, Rubin K, Heldin P. Expression of hyaluronan synthase 2 or hyaluronidase 1 differentially affect the growth rate of transplantable colon carcinoma cell tumors Int J Cancer 2002; 102(3):212-9.
• ¹¹⁸
  Padavattan S, Schirmer T, Schmidt M, Akdis C, Valenta R, Mittermann I et al..Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific fab J Mol Biol 2007; 368(3):742-52.
• ¹¹⁹
  Valenta R, Niederberger V. Recombinant allergens for immunotherapy J Allergy Clin Immunol 2007; 119(4):826-30.
• ¹²⁰
  Cromwell O, Hafner D, Nandy A. Recombinant allergens for specific immunotherapy J Allergy Clin Immunol 2011; 127(4):865-72.
• ¹²¹
  Rolland JM, Gardner LM, O’Hehir RE. Allergen-related approaches to immunotherapyPharmacol Ther 2009; 121(3):273-84.
• ¹²²
  Kohler J, Blank S, Mueller S, Bantleon F, Frick M, Huss-Marp J et al.. Component resolution reveals additional major allergens in patients with honeybee venom allergy J Allergy Clin Immunol 2014; 133(5):1383-9.
• ¹²³
  Justo Jacomini DL, Gomes Moreira SM, Campos Pereira FD, Zollner RL, Brochetto Braga MR.Reactivity of IgE to the allergen hyaluronidase from Polybia paulista (Hymenoptera, Vespidae) venom Toxicon 2014; 82:104-11.
• ¹²⁴
  Balzer L, Pennino D, Blank S, Seismann H, Darsow U, Schnedler M et al.. Basophil activation test using recombinant allergens: highly specific diagnostic method complementing routine tests in wasp venom allergy PLoS ONE 2014; 9(10): Article ID E108619
• ¹²⁵
  Mittermann I, Zidarn M, Silar M, Markovic-Housley Z, Aberer W, Korosec P et al..Recombinant allergen-based IgE testing to distinguish bee and wasp allergy J Allergy Clin Immunol 2010; 125(6):1300-7.
• ¹²⁶
  Valenta R, Niespodziana K, Focke-Tejkl M, Marth K, Huber H, Neubauer A et al..Recombinant allergens: What does the future hold? J Allergy Clin Immunol 2011;127(4):860-4.
• ¹²⁷
  Bitencourt CS, Pereira PA, Ramos SG, Sampaio SV, Arantes EC, Aronoff DM et al..Hyaluronidase recruits mesenchymal-like cells to the lung and ameliorates fibrosisFibrogenesis Tissue Repair 2011; 4(1):3.
• ¹²⁸
  Bitencourt CS, Gelfuso GM, Pereira PA, Assis PA, Tefé-Silva C, Ramos SG et al..Hyaluronidase-loaded PLGA microparticles as a new strategy for the treatment of pulmonary fibrosis Tissue Eng Part A 2015; 1–2:246-56.
• ¹²⁹
  Sunagar K, Undheim EAB, Chan AHC, Koludarov I, Munoz-Gomez SA, Antunes A et al..Evolution stings: the origin and diversification of scorpion toxin peptide scaffoldsToxins 2013; 5(12):2456-87.
• ¹³⁰
  Morgenstern D, Rohde BH, King GF, Tal T, Sher D, Zlotkin E. The tale of a resting gland: transcriptome of a replete venom gland from the scorpion Hottentotta judaicusToxicon 2011; 57(5):695-703.
• ¹³¹
  de Oliveira UC, Candido DM, Dorce VA. Junqueira-de-Azevedo I de L. The transcriptome recipe for the venom cocktail of Tityus bahiensis scorpion Toxicon 2015; 95:52-61.
• ¹³²
  Keeling CI, Yuen MM, Liao NY, Docking TR, Chan SK, Taylor GA et al.. Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pestGenome Biol 2013; 14(3):R27.
• ¹³³
  Richards S, Gibbs RA, Weinstock GM, Brown SJ, Denell R, Beeman RW et al.. The genome of the model beetle and pest Tribolium castaneum Nature 2008; 452(7190):949-55.
• ¹³⁴
  Ma D, Li Y, Dong J, An S, Wang Y, Liu C et al.. Purification and characterization of two new allergens from the salivary glands of the horsefly Tabanus yao Allergy 2011;66(1):101-9.
• ¹³⁵
  Russell CL, Heesom KJ, Arthur CJ, Helps CR, Mellor PS, Day MJ et al.. Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus Insect Mol Biol 2009; 18(3):383-93.
• ¹³⁶
  Campbell CL, Vandyke KA, Letchworth GJ, Drolet BS, Hanekamp T, Wilson WC. Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae) Insect Mol Biol 2005; 14(2):121-36.
• ¹³⁷
  Schaffartzik A, Marti E, Torsteinsdottir S, Mellor PS, Crameri R, Rhyner C. Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity Vet Immunol Immunopathol 2011; 139(2–4):200-9.
• ¹³⁸
  van der Meide NM, Roders N, van Oldruitenborgh-Oosterbaan MMS, Schaap PJ, van Oers MM, Leibold W et al.. Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses Vet Immunol Immunophatol 2013; 153(3–4):227-39.
• ¹³⁹
  Chagas AC, Calvo E, Pimenta PF, Ribeiro JM. An insight into the sialome of Simulium guianense (Diptera:Simuliidae), the main vector of River Blindness Disease in BrazilBMC Genomics 2011; 12:612.
• ¹⁴⁰
  Ribeiro JMC, Chagas AC, Pham VM, Lounibos LP, Calvo E. An insight into the sialome of the frog biting fly, Corethrella appendiculata Insect Biochem Molec Biol 2013; 44:23-32.
• ¹⁴¹
  Nene V, Wortman JR, Lawson D, Haas B, Kodira C, Tu ZJ et al.. Genome sequence of Aedes aegypti, a major arbovirus vector Science 2007; 316(5832):1718-23.
• ¹⁴²
  Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR et al.. The genome sequence of the malaria mosquito Anopheles gambiae Science 2002;298(5591):129-49.
• ¹⁴³
  Marinotti O, Cerqueira GC, de Almeida LG, Ferro MI, Loreto EL, Zaha A et al.. The genome of Anopheles darlingi, the main neotropical malaria vector Nucleic Acids Res 2013;41(15):7387-400.
• ¹⁴⁴
  Chagas AC, Calvo E, Rios-Velasquez CM, Pessoa FAC, Medeiros JF, Ribeiro JMC. A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes BMC Genomics 2013; 14:875.
• ¹⁴⁵
  Hostomska J, Volfova V, Mu J, Garfield M, Rohousova I, Volf P et al.. Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus BMC Genomics 2009;10:282.
• ¹⁴⁶
  Rohousova I, Subrahmanyam S, Volfova V, Mu J, Volf P, Valenzuela JG et al.. Salivary gland transcriptomes and proteomes of Phlebotomus tobbi and Phlebotomus sergenti, vectors of leishmaniasis PLoS Negl Trop Dis 2012; 6(5): Article ID e1660
• ¹⁴⁷
  Vlkova M, Sima M, Rohousova I, Kostalova T, Sumova P, Volfova V et al.. Comparative analysis of salivary gland transcriptomes of Phlebotomus orientalis sand flies from endemic and non-endemic foci of visceral leishmaniasis PLoS Negl Trop Dis 2014;8(2): Article ID e2709
• ¹⁴⁸
  Nygaard S, Zhang G, Schiott M, Li C, Wurm Y, Hu H et al.. The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming Genome Res 2011; 21(8):1339-48.
• ¹⁴⁹
  Suen G, Teiling C, Li L, Holt C, Abouheif E, Bornberg-Bauer E et al.. The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle PLoS Genet 2011; 7(2): Article ID e1002007
• ¹⁵⁰
  Bonasio R, Zhang G, Ye C, Mutti NS, Fang X, Qin N et al.. Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator Science 2010;329(5995):1068-71.
• ¹⁵¹
  Oxley PR, Ji L, Fetter-Pruneda I, McKenzie SK, Li C, Hu H et al.. The genome of the clonal raider ant Cerapachys biroi Curr Biol 2014; 24(4):451-8.
• ¹⁵²
  Wurm Y, Wang J, Riba-Grognuz O, Corona M, Nygaard S, Hunt BG et al.. The genome of the fire ant Solenopsis invicta Proc Natl Acad Sci U S A 2011; 108(14):5679-84.
• ¹⁵³
  Baek JH, Lee SH. Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae) Toxicon 2010; 55(6):1147-56.
• ¹⁵⁴
  Baek JH, Woo TH, Kim CB, Park JH, Kim H, Lee S et al.. Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae) Arch Insect Biochem Physiol 2009; 71(4):205-22.
• ¹⁵⁵
  An S, Chen L, Wei JF, Yang X, Ma D, Xu X et al.. Purification and characterization of two new allergens from the venom of Vespa magnifica PLoS ONE 2012; 7(2): Article ID e31920
• ¹⁵⁶
  Terrapon N, Li C, Robertson HM, Ji L, Meng X, Booth W et al.. Molecular traces of alternative social organization in a termite genome Nat Commun 2014; 5:3636.
• ¹⁵⁷
  Zhan S, Merlin C, Boore JL, Reppert SM. The monarch butterfly genome yields insights into long-distance migration Cell 2011; 147(5):1171-85.
• ¹⁵⁸
  Xia Q, Wang J, Zhou Z, Li R, Fan W, Cheng D et al.. The genome of a lepidopteran model insect, the silkworm Bombyx mori Insect Biochem Mol Biol 2008; 38(12):1036-45.
• ¹⁵⁹
  Kirkness EF, Haas BJ, Sun W, Braig HR, Perotti MA, Clark JM et al.. Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle Proc Natl Acad Sci U S A 2010; 107(27):12168-73.

Abbreviations

• 3D  Three-dimensional
• CHO  Chinese hamster ovary
• ECM  Extracellular matrix
• GlcNAc  N-acetyl-β-D-glucosamine
• GlcUA  β-D-glucuronic acid
• HYAL-MP  Microparticles loaded with hyaluronidase
• MPs  Microparticles
• PDB  Protein data bank
• PLGA  Poly (D,L-lactide-co-glycolide)
• rHuPH20  Recombinant human PH-20 hyaluronidase

Publication Dates

• Publication in this collection
  2015

History

• Received
  2 Feb 2015
• Reviewed
  8 Oct 2015
• Accepted
  22 Oct 2015