Hyaluronidase activity in leeches (Hirudinea)

Comparative Biochemistry and Physiology Part B 124 (1999) 319 – 326 www.elsevier.com/locate/cbpb

Hyaluronidase activity in leeches (Hirudinea) Peter Hovingh *, Alfred Linker Veterans Administration Hospital, 151 E, Salt Lake City, UT 84148, USA Received 1 April 1999; received in revised form 27 July 1999; accepted 3 August 1999

Abstract The leech hyaluronoglucuronidase (hyaluronidase I) was identified in Erpobdellidae (Nephelopsis obscura and Erpobdella punctata) and Glossiphoniidae (Desserobdella picta) and historically described from Hirudinidae (Hirudo medicinalis). A second leech hyaluronidase (hyaluronidase II) which hydrolyzed only a few bonds to form hyaluronan oligosaccharides larger than 6500 Da, was found in Glossiphoniidae (Helobdella stagnalis, Glossiphonia complanata, Placobdella ornata, and Theromyzon sp.) and in Haemopidae (Haemopis marmorata). The distribution of the two hyaluronidases in leech occurred in both orders (Arhynchobdellida and Rhynchobdellida) and in macrophagous and haematophagous feeding types whereas the liquidosomatophagous leeches only had hyaluronidase II. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Erpobdellidae; Glossiphoniidae; Haemopidae; Hirudinidae; Hyaluronan; Hyaluronidase; Leech; Comparative biochemistry

1. Introduction Hyaluronidase consists of several types of enzymes that degrade hyaluronan. Bacterial hyaluronidase (hyaluronate lyase) degrades hyaluronan by a b-elimination reaction in which an a, b unsaturated uronic acid linked to N-acetylglucosamine is formed. Most invertebrate and vertebrate hyaluronidases (hyaluronoglucosamidase) are hydrolases forming products varying from disaccharides to oligosaccharides with glucuronic acid at the non-reducing end and Nacetylglucosamine at the reducing end. A third hyaluronidase (hyaluronoglucuronidase) is a hydrolase found in leeches and forms tetrasaccharides and large oligosaccharides with N-acetylglucosamine at the nonreducing end and glucuronate at the reducing end [8,13]. The leech hyaluronidase has been purified from the leeches Hirudo medicinalis Linnaeus 1758, H. nipponia Whitman 1886, and Poecilobdella hubeiensis Yang 1980 [30,32] and the products of this enzyme were characterized [16]. More recently, a heparanase from mammalian sources also has a specificity similar to hyaluronoglucuronidase in that the oligosaccharide * Corresponding author. Tel.: +1-801-5821565, ext. 1495; fax: +1-801-5839624. E-mail address: [email protected] (P. Hovingh)

products contain glucuronate at the reducing end in products from the glycosaminoglycan heparin and heparan sulfate [7]. The above mentioned leeches are all haematophagous and belong to the same leech family Hirudinidae (Order Arhynchobdellida). In this paper we have examined the hyaluronidase activity in leeches of the Order Rhynchobdellida: Glossiphoniidae: Glossiphonia complanata Linnaeus 1758, Theromyzon sp., Placobdella ornata Verrill 1872, Desserobdella picta Verrill 1872, and Helobdella stagnalis Linnaeus 1758, and of the Order Arhynchobdellida: Erpobdellidae: N. obscura Verrill 1872 and Erpobdella punctata Leidy 1870; and Haemopidae: Haemopis marmorata Say 1824. These leeches are predaceous and liquidosomatophagous (Glossiphonia and Helobdella), haematophagous (Placobdella, Theromzyon, and Desserobdella), and predaceous and macrophagous (Nephelopsis, Erpobdella, and Haemopis) [24]. The diet of these leeches is variable, with the leech D. picta feeding on amphibians [1,27] and in our specimens, on the tiger salamander Ambystoma tigrinum; P. ornata feeding on turtles [18] and in this source material mammals (turtles are not indigenous in Utah); Theromyzon feeding on birds [4]; G. complanata feeding on Mollusca, Oligochaeta, Crustacea, and aquatic insects [9,28,31]; H. stagnalis, N. obscura, and E. punctata

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feeding on aquatic insects, Crustaceans, and Oligochaeta [2,5,6,28,31]; H. marmorata feeding on both macroinvertebrates and small vertebrates (Hovingh, observations). The hyaluronidase activity from these leeches is compared to the hyaluronidase from H. medicinalis. We also examined the substrate specificity of Hirudo hyaluronoglucuronidase to determine if it could act on other polysaccharides that contain uronic acid.

difficult to determine. Change in molecular size was utilized to examine substrate specificity. Shark cartilage chondroitin sulfate (8% non-sulfated disaccharide) from Sigma, heparan sulfate (9% sulfate) [14] as well as three undescribed low sulfated polysaccharides from the zebra mussel Dreissena polymorpha (from Michigan) with 1.6% uronate, from the Tubifex (San Francisco Bay Brands, Inc.) with 10% uronate, and from N. obscura with 27.1% uronate all eluted near the void volume on Sepharose 6B.

2. Materials and methods

2.3. Chromatography

2.1. Materials

Sepharose 2B column (56× 1 cm) was eluted with 0.15 M NaCl; Sepharose 6B was eluted with 0.04 M urea; Sephadex G-50 (48× 1 cm) was eluted with 0.075 M NaCl; and Sephadex G-25 fine (61 × 1 cm) was used for desalting and elution occurred with water. Paper chromatograms were developed with a descending solvent system: (1) n-butanol/acetic acid/water (50/15/35 v/v) and with (2) ethyl acetate/pyridine/water (80/2.5/1 v/v).

Bovine liver b-glucuronidase (Sigma G-0501), Aspergillus b-N-acetylhexosaminidase (Sigma A-7708), bovine testes hyaluronidase (Sigma H-3884, 990 units per mg, EC 3.2.1.35), hyaluronate (Sigma H-1876), p-nitrophenyl-N-acetylglucosamine (CalBiochem 487052), phenolphthalein-glucuronide (CalBiochem 51635) were commercial products. Leech hyaluronidase (EC 3.2.1.36) was prepared from H. medicinalis obtained from Biopharm Leeches™ (The Gadsden House, 329 East Bay Street, Charleston, South Carolina 29401). Haemopis was obtained from central Minnesota and all other leeches were obtained from local environments of abundant populations near Salt Lake City, Utah. Column support materials were obtained from Pharmacia (Sepharose 2B, Sepharose 6B, Sephadex G-50, and Sephadex G-25) and paper chromatography from Whatman (1 Chr). Tetrasaccharide from testicular hyaluronidase products of hyaluronan was prepared as described [16].

2.2. Analysis Viscosity was measured with a Cannon Ubbelohde semi micro viscometer in a water bath at 37°C with readings at 0, 15, 30, 60, and 120 min with 10 ml of enzyme (20 or 200 mg of enzyme) added to 1 ml 0.1 N NaAcetate, pH 6.0 with 2 mg hyaluronan. Analytical procedures involved reducing sugar [22,25], N-acetylglucosamine [12,13,23], uronic acid [3], hexosamine [10], reducing sugar K-borohydride reduction [11], and protein analysis by the BioRad Assay using bovine serum albumin (Sigma A-3350) as a standard. Hyaluronidase was assayed by viscometry and by reducing sugar using glucose as a control. The reducing sugar sensitivity was confounded by a high background reading at zero time. A more sensitive assay followed the change in molecular size by column chromatography of Sepharose 2B. This column chromatography assay could not ascribe activity to numerical form as the changes in molecular size produced a broad range of sizes making the number of carbohydrate linkages

2.4. Hyaluronidase preparation Leeches were cooled or frozen at −70°C and cut into a head fraction (anterior to the male gonophore) and a crop fraction (representing the anterior 50% of the headless body), with the intestine (posterior region) not analyzed. Each fraction was ground with sand by pestle and mortar in 0.15 M NaCl and extracted overnight in the cold. These samples were centrifuged (1500× g for 15 min) and the supernatant fluid was brought to 80% ammonium sulfate and equilibrated overnight. These samples were then centrifuged (1500× g for 15 min) and the precipitate was taken up in 0.1 M NaAcetate, pH 6.0, dialyzed overnight against water in the cold, and lyophilized. The methods largely follow the initial preparation of Yuki and Fishman [32] and are crude enzyme preparations. A unit is defined here as the difference in viscometric time in minutes between zero and 15 min of incubation at 37°C. All the comparative analysis is based on the dry weight preparation, but specific activity and the amount of protein in each of the assays can be determined from Table 1 from the protein analysis. All experiments were performed on pooled samples as represented in Table 1 and Table 2.

2.5. Hyaluronidase nomenclature This study found two leech hyaluronidases. In what follows, leech hyaluronidase I is the traditional leech hyaluronidase (hyaluronoglucuronidase) [13,16,30,32]. Leech hyaluronidase II is reported here for the first time and it hydrolyzes hyaluronan to large oligosaccha-

P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 124 (1999) 319–326

rides and not to tetrasaccharides. It is unknown whether this leech hyaluronidase II is a hyaluronoglucuronidase or a hyaluronoglucosamidase.

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the other leeches had low values, although Haemopis had intermediate values of units per leech.

3.3. Analysis of hyaluronidase products 3. Results

3.1. Comparison of Hirudo hyaluronidase with bo6ine testicular hyaluronidase Fig. 1 shows the rates of hydrolysis with bovine testicular hyaluronidase at three (15, 60, and 120 min) time intervals by viscometry. The hyaluronidase activity was proportional to enzymic units between 0.5 and 5.0 mg enzyme. The comparison of reducing sugar formation (Fig. 2A) and viscometric rates (Fig. 2B) are shown for Hirudo and bovine testicular hyaluronidase. The Hirudo head preparation is ten times more active than the bovine testicular hyaluronidase preparation used.

3.2. Comparison of hyaluronidase in leeches Table 1 shows the enzyme yield of hyaluronidase in the head and crop of nine species of leeches. The head (except from Desserobdella and Haemopis) contained the most active preparation (units per weight) whereas the crop (except Hirudo) contained the most activity (total units). Table 2 summarizes the viscometry activity of the hyaluronidase of the nine species of leeches. Hirudo contained, by an order of magnitude, more total units, higher activity (units per weight), and more units per leech than all the other species. Erpobdella, Nephelopsis, and Desserobdella had intermediate values and

The size of the hyaluronidase products were determined by two protocols. In the first experiment, the samples were hydrolyzed at levels of 4 mg hyaluronan and 3 mg dry weight leech preparation in 1 ml. After aliquots were removed for reducing sugar analysis, the sample was placed on Separose 2B (Fig. 3). The head preparation from Helobdella, Glossiphonia and Placobdella degraded hyaluronan very poorly, leaving material that eluted in the void volume. All the other leech preparations degraded hyaluronan to products that eluted near the included volume of the column. In the second experiment, 10 mg hyaluronan was digested with 3 mg dry weight of leech preparation, followed by another 4 mg dry weight of leech preparation from the most active region of each leech. The Hirudo hydrolysis was done with only 3 mg dry weight leech preparation and no second addition. This hydrolyzed material was eluted from Sephadex G-50 (Fig. 4). The void volume material (at least 6500 molecular weight, see [14,15] was further digested with some 3–4 mg enzyme and re-eluted from Sephadex G-50. Under these conditions Glossiphonia, Haemopis, and Placobdella hyaluronan products largely remained in the void volume, suggesting a product size between 6500 and 14 000 Da. Helobdella and Theromyzon was further digested, but did not form any products of tetrasaccharide size. Desserobdella, Nephelopsis, and Erpobdella preparations further degraded the hyaluronan products to form tetrasaccharides.

Table 1 Hyaluronidase activity in leech crop and heada

Helobdella head crop Glossiphonia head crop Theromyzon head and crop Placobdella head crop Desseobdella head crop Erpobdella head crop Nephelopsis head crop Haemopis head crop Hirudo head crop

Enzyme yield (mg)

Percent protein

Total units

Total units per g wet weight

7 14 16 44 27 10 32 24 134 27 64 21 59 20 97 22 285

29 64 36 50 94 36 50 30 92 68 100 41 74 94 90 32 100

13 71 30 123 26 46 58 53 509 245 553 636 794 16 117 13876 857

55 32 58 28 90 102 29 111 117 278 205 722 325 27 38 42048 201

Activity determined by viscometry with 2 mg/ml hyaluronate in 0.1 N Acetate, pH 6 and 50 mg bovine testicular hyaluronidase enzyme at 37°C. Hirudo enzyme was assayed at 5 mg. A unit is defined as initial viscosity time less the viscosity time at 15 min and is equivalent to 10 N.F. units of bovine testicular hyaluronidase. a

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Table 2 Hyaluronidase yield from leechesa

Helobdella Glossiphonia Theromyzon Placobdella Desseobdella Erpobdella Nephelopsis Haemopis Hirudo a

No. of leeches

Total wet weight (g)

Total units

Total units per g wet weight

Total units per leech

400 75 6 18 55 23 17 2 1

3.5 6.2 0.8 4.1 6.6 6.2 5.2 6.6 7.5

84 153 26 104 562 799 1430 134 14733

24 24 32 25 85 128 275 20 1964

0.2 2.1 4.4 5.8 10.2 34.8 84.2 67.1 14733

Activity determined by viscometry as described in Table 1.

The products from the experiment represented in Fig. 4 were desalted on Sephadex G-25 for further analysis. Paper chromatography showed tetrasaccharide, hexasaccharide, and octasaccharide products from Hirudo, Erpobdella, Nephelopsis, and Desserobdella preparations, and these products were not observed in Glossiphonia, Helobdella, Theromyzon, and Placobdella preparations (not shown). The tetrasaccharide products from the leeches migrated more slowly than the tetrasaccharide obtained from hyaluronate by testicular hyaluronidase as expected [16]. The analysis of the products is shown in Table 3. The N-acetylglucosamine (assay determines monosaccharides and N-acetylglucosamine at the reducing end of testicular hyaluronidase derived oligosaccharides) was found in hyaluronan products of testicular hyaluronidase and hyaluronan with molar ratio of reducing end N-acetylglucosamine to reducing sugar greater than 0.36. The leeches had trace levels of N-acetylglucosamine with the molar ratio less than 0.24 and Hirudo having a ratio of 0.01 suggesting that N-acetyl glucosamine and glucuronate were at the reducing end. No N-acetylglucosamine was detected in a Glossiphonia enzyme digestion without hyaluronan. The products in Table 3 were reduced with alkaline borohydride, resulting in a total loss of the N-acetylglucosamine and reducing sugar (not shown). Treatment of the hyaluronan products from Hirudo, Nephelopsis, and Desserobdella with bovine liver b-glucuronidase (which also contained bN-acetylglucosaminidase) for 2 h resulted in monosaccharide N-acetylglucosamine products on paper chromatography. N-acetylglucosamine was not obtained from control treatment of the tetrasaccharide and the testicular hyaluronidase generated oligosaccharide products. Longer term digestions under these conditions resulted in N-acetylglucosamine and uronate products from all the preparations. The Aspergillus hexosaminidase did not act on the products. Although leech hyaluronidase I does not form products that have N-acetylglucosamine at the reducing end (note the Hirudo products in Table 3), the crude prepa-

ration utilized in these studies, with secondary reactions, may account for the presence of N-acetylglucosamine at the reducing end of the hyaluronan products.

3.4. Substrate specificity The Hirudo preparation was tested to determine if the leech hyaluronidase I would act on other substrates as might be encountered in the digestive processes of the leech. This preparation did not degrade heparan sulfate, chondroitin sulfate and three undescribed uronate-containing polysaccharides from the zebra mussel, tubifex, or from the leech Nephelopis.

4. Discussion The experimental objective in these studies on leech hyaluronidase was to detect very low activities of enzymes, based on our earlier unpublished work on N. obscura which failed to detect any hyaluronidase activity. Since Nephelopsis was not haematophagous, the lack of hyaluronidase in this species was expected, albeit the methods used in the paper did find activity.

Fig. 1. Viscometry times (0 time minus time at 15, 60, and 120 min of incubation) versus protein concentration of bovine testicular hyaluronidase. Hyaluronate (2 mg) and 10 ml enzyme were in 1 ml buffer.

P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 124 (1999) 319–326

Fig. 2. Comparative kinetics of Hirudo and testicular hyaluronidase. (A) Formation of reducing sugar per 400 mg of hyaluronate in an assay with 4 mg hyaluronate per ml and Hirudo enzyme at 20 mg (square); bovine testicular enzyme at 200 mg (solid circle); and at 100 mg (open circle) per ml. (B) Viscometry times (same conditions as Fig. 1) versus incubation time with Hirudo hyaluronidase at 5 mg (square); testicular enzyme at 50 mg (solid circle); and at 15 mg (open circle) per ml.

Also, liquidosomatophagous feeding leeches may well utilize hyaluronidase to assist in the breakdown of the tissue of the prey. Since leeches have evolved at least two distinct blood feeding procedures in the two distinct branches of leech evolution (Orders Arhynchobdellida and Rhynchobdellida) [26], it was essential to examine the haematophagous leeches in species from different families. One factor not controlled in our study was the state of feeding (active or inactive). Total activity could be rather variable depending on the feeding state of the leech [24], suggesting a more thorough study of hyaluronidase activity in leeches. The presence of hyaluronidase activity in the head or crop is correlated with the location of the salivary glands. The ‘head’ in these studies represents the region anterior to the male gonopore in segments XI–XII. Thus H. medicinalis salivary glands are found in segments VI–XII [24] and the hyaluronidase is largely found in the head region. G. complanata salivary glands are found in segments XI – XVIII [24]; P. ornata in segments VIII–X (anterior glands) and IX – XI (posterior glands) [19]; and Desserobdella picta in segments VII – XV [19]. These Glossiphoniidae hyaluronidase was largely found in the ‘crop’ region, suggesting an association with the salivary glands. Additional studies using leech hyaluronidase I and II antibodies and histology would contribute to the tissue location of hyaluronidase in leeches. Although endosymbiotic prokaryotes may occur in the ‘head’ and ‘crop’ region, all known bacteria hyaluronidase degrades hyaluronan to disaccharides. In the leeches examined, Desserobdella picta (Glossiphoniidae), E. punctata and N. obscura (Erpobdellidae), and H. medicinalis (Hirudinidae) hyaluronidase conform to the properties of leech hyaluronidase I in that tetrasaccharides are formed with N-acetylglucosamine at the non-reducing end. The low molecular weight products could be further metabolized as a nutrient.

323

P. ornata, G. complanata, H. stagnalis, and Theromyzon sp. (Glossiphoniidae) and H. marmorata (Haemopidae) hyaluronidase preparations did not form the tetrasaccharides (Fig. 4), suggesting a different hyaluronidase in these leeches. This hyaluronidase II may likewise be hyaluronoglucuronidase based on the low reducing end N-acetylglucosamine to reducing sugar ratio. However, the linkage specificity of hyaluronidase II is still considered unknown. This second enzyme would only be involved in what was once referred to as the ‘spreading factor’, decreasing the tissue viscosity to allow faster movement of nutrients from the host to the leech. Alternatively, hyaluronidase II activity may be secondary to other activities of the enzyme, that is, having substrate specificity beyond hyaluronan. A summary of the distribution of the two hyaluronidases and feeding behavior is found in Table 4.

Fig. 3. Elution patterns of hyaluronate products by hyaluronidase preparations from leeches. The Sepharose 2B column void volume was 18 ml and the included volume was 54 ml. Hyaluronate (4 mg) and dry weight leech preparations (3 mg) in 1 ml were incubated for 24 h. Large solid circle, head preparations; small solid circle, crop preparations; open circle, hyaluronate without treatment. (A) Hirudo; (B) Glossiphonia; (C) Desserobdella; (D) Nephelopsis; (E) Helobdella; (F) Haemopis; (G) Erpobdella; (H), Placobdella; and (I) Theromyzon (head and crop combined).

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Fig. 4. Elution patterns of hyaluronate products by hyaluronidase preparations from leeches. The Sephadex G-50 column void volume was 13 ml and the included volume was 37 ml. The head preparations of Hirudo, Haemopis, Nephelopsis, and Desserobdella; the crop preparations of Helobdella, Erpobdella, Glossiphonia, and Placobdella; and both head and crop preparation of Theromyzon were utilized. Hyaluronate (10 mg) and dry weight leech preparation (3 mg) was followed after 24 h incubation with 4 mg additional dry weight leech preparation and a second 24-h incubation (line represention). Only the 3 mg dry weight leech preparation and 24-h incubation occurred with Hirudo. The hyaluronate products were desalted and retreated with 3 – 4 mg additional dry weight leech preparation for 24 h and reapplied to the column (line with solid circles). (A) Hirudo; (B) Glossiphonia; (C) Desserobdella; (D) Nephelopsis; (E) Helobdella; (F) Haemopis; (G) Erpobdella; (H) Placobdella; and (I) Theromyzon (head and crop combined).

Blood feeding behavior in leeches has divergent processes and involves the antithrombin hirudin in the family Hirudinidae, the fibrinolysis hementin in Haementeria ghilianii de Filippi 1849, and the plasminogen-activator in Haementeria depressa Blanchard 1849 of the family Glossiphoniidae [24,26,29]. Thus different physiological mechanisms have evolved in the anticoagulant phase of the hematophagia. Leech hyaluronidase I is found in both Hirudinidae: Hirudo and Poecilobdella [13,30] and Glossiphoniidae: Desserobdella (this paper). Leech hyaluronidase II is found in P. ornata and Theromyzon suggesting, as in the case of anticoagulants, that Glossiphoniidae members also have different physiological mechanisms involving hyaluronidase with blood-feeding on vertebrates (Table 4).

The function of leech hyaluronidase in the diverse group of leeches studied in this paper may suggest that the digestion processes on the diverse group of prey are not species-specific or even class and phyla specific. H. stagnalis feeds on Annelida, Arthropoda, and Mollusca fauna [6,28,31], but has been noted on vertebrates [17,21] although critical serological proofs of feeding has not occurred for these events. Although P. ornata has generally utilized turtle and mammalian blood, it can feed on Mollusca [20]. The most specific host-feeding leech in this study is Desserobdella picta, an amphibian blood feeding leech that may also feed on turtles [27]. The diverse feeding pattern of leeches was the principal motivation to study hyaluronidase in leeches. The high activity of leech hyaluronidase I in at least three species of Hirudinidae and the low activity in Desserobdella picta might suggest a response to homeothermic (mammals) and poikilothermic (amphibians) prey, respectively. However, the presence of leech hyaluronidase I in N. obscura and E. punctata would suggest a use in macrophagous feeding on invertebrates. Its presence in Erpobdellidae, Hirudinidae, and Glossiphoniidae, on the other hand, would suggest an origin associated with early evolution of leeches. The presence of hyaluronidase II in Glossiphoniidae (Helobdella, Glossiphonia, Theromyzon, and Placobdella) and in Haemopidae (the macrophagous Haemopis) confounds the evolutionary tree as well as the relationship of hyaluronidase to haematophagous, macrophagous, and liquidosomatophagous feeding patterns. The overlapping of the Table 3 Hyaluronidase products from leech preparationsa Percent uronate

Ratios

Helobdella (a) Helobdella (b) Glossiphonia (a) Glossiphonia (b) Theromyzon (a) Theromyzon (b) Placobdella Desserobdella Erpobdella Nephelopsis Haemopis Hirudo

43 42 4 37 31 31 43 44 20 45 24 42

1.00: 1.00: 1.00: 1.00: 1.00: 1.00: 1.00: 1.00: 1.00: 1.00: 1.00: 1.00:

Total testicular enzyme products Testicular tetrasaccharide Hyaluronate control

41

1.00: 0.86: 0.67: 0.24

56 28

1.00: 0.69: 0.97: 0.52 1.00: 0.93: 0.07: 0.03

0.82: 0.45: 0.87: 0.47: 0.82: 1.02: 0.78: 0.78: 1.01: 0.75: 1.25: 0.59:

0.23: 0.50: 0.17: 0.42: 0.31: 0.38: 0.13: 0.47: 0.54: 0.54: 0.17: 0.77:

0.05 0.14 0.04 0.05 0.01 0.01 0.01 0.02 0.06 0.01 0.03 0.01

a Molar ratio of total uronate, total glucosamine, reducing sugar, and reducing end N-acetyl glucosamine to total uronic acid. (a) is the large molecular size of the elution and (b) is the smaller size of the elution.

P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 124 (1999) 319–326 Table 4 Summary of resultsa [2] Feeding Group

Food type

Hyaluronidase type [3]

Rhynchobdellida: Glossiphoniidae Helobdella stagnalis Glossiphonia complanata Desserobdella picta Theromyzon sp Placobdella ornata

[4] L L

i i

II II

H H H

a b t,m

I II II

[5]

Arhynchobdellida: Erpobdelliformes Erpobdellidae Erpobdella punctata Nephelopsis obscura

M M

i i

I I

Arhnchobdellida: Hirudiniformes Haemopidae Haemopis marmorata

M

i

II

Hirudinidae Hirudo medicinalis

H

a,m

I

[6]

[7]

a Feeding type: liquidosomatophagous (L); haematophagous (H); and macrophagous (M). Food type: invertebrates (i); amphibian (a); turtle (t); birds (b); mammals (m). Hyaluronidase type: Leech hyaluronidase I, leech hyaluronidase II.

two hyaluronidases with the two orders of leeches suggest an early origin of both hyaluronidases (Table 4). However, as their properties seem to be quite different, it is unlikely that hyaluronidase II merely represents an activity left from an ancestor of the class of leeches. It must also be considered that though degradation by hyaluronidase II is limited, it may still be adequate for any biological function required.

Acknowledgements We greatly thank Roy T. Sawyer for donating H. medicinalis for this work, without which this effort would be rather meaningless. We also thank William Moser for prodding this research forward at a time when both leeches and hyaluronidase were relevant to the authors and Mike Piepkorn for reviewing the paper. These studies were supported in part by the U.S. Public Health Service grant (no. AR 21557) from the National Institutes of Health Human Services and by the Office of Research and Development, Medical Research Service, Department of Veteran Affairs.

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