Biosci, Biotechnol. Biochem., 62 (3), 459-463, 1998 IS3A A Very-high-density Lipoprotein with Clotting Ability from Hemolymph of Sand Crayfish, Ibacus ciliatus Masaharu Komarsu* and Seiichi Anpo** “Laboratory of Food Chemistry and **Lipoprotein Research Laboratory, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890, Japan Received August 11, 1997 A very-high-density lipoprotein (VHDL) with a density of 1.27-1.29 g/ml was the most abundant lipoprotein in the hemolymph of the sand crayfish Zbacus cillatus. The VHDL isolated by a density gradient ultracentrifugation consisted of 94% protein and 6% lipid reflecting its high density, and phospholipid was a predominant lipid compo- nent. The VHDL had an apolipoprotein of molecular mass 195 kDa and its N-terminal amino acid sequence was iden- tified as follows: LQPGLEYQYRYNGRVAA. This se ‘quence was similar to those of clotting proteins from the spiny lobster Panulirus interruptus and the freshwater crayfish Pacifastacus leniusculus. Transglutaminase and Cx#* also induced the VHDL to clot. Considering large amounts of VHDL in the hemolymph of sand crayfish, the VHDL not only functions as lipid carrier but plays an im- portant role in the defense process of crustacean, Key words: clotting protein; crustacea; fibrinogen; sand crayfish; very-high-density lipoprotein In the circulatory fluid of animals including crustacea, most lipids are complexed with protein in the form of lipoproteins. Lipoproteins of all animals func- tion as major carriers of lipid and other hydrophobic compounds.” The vertebrate lipoproteins consist of very-low-density, low-density, and high-density lipoprotein (HDL), while HDL as the main lipoprotein is isolated from the hemolymph of invertebrates such as. insects’ and crustacea.*” The insect HDL called lipophorin is apparently different from the counterpart of crustacea in structure and function, serving as a reusa- ble shuttle of diacylglycerol. The crustacean HDL. reported to date is characterized by a high level of phos- pholipid (PL) and a single apolipoprotein with molecu- lar mass (M,) of 100-120 KDa.™ ‘We have recently characterized the lipoprotein fea- tures in the hemolymph of some crustacea, in which very-high-density lipoprotein (VHDL) was newly isolat- ed from the hemolymph, as well as HDL." Although PL was the main lipid component in both VHDL and HDL, the lipid content of VHDL was much lower than that of HDL, reflecting its high density (d) of 1.27-1.29 g/ml. Considering the very low level of lipid in VHDL, VHDL might play other roles than lipid carriers. Human HDL and insect lipophorin function not only as lipid carriers but as vital defense factors such as f- 1,3-glucan and lipopolysaccharide binding proteins.'""" Hall er al. recently isolated HDL and VHDL from the hemolymph of a crustacean, the freshwater crayfish Pacifastacus leniusculus.' Interestingly, the HDL and VHDL were identified as f-1,3-glucan binding pro- tein" and clotting protein," respectively, which were important components in the defense reactions of crayfish. The f-1,3-glucan is a trigger for the phenol oxi dase cascade system in invertebrates, by which microor- ganisms are enclosed in melanin. The clotting reaction in crustacea occurs by both cellular and humoral fac- tors, which are the substrates for Ca**-dependent trans- glutaminase (TGase) derived from the hemocyte."” TGase catalyzes the formation of cross-linked protein aggregates between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another protein. The clotting protein from the hemo- lymph of crayfish consists of two identical subunits with M, 210 kDa, covalently associated by disulfide bonds. The clotting protein called fibrinogen, the subunits of which are similar to their counterparts in crayfish, is also isolated from the hemolymph of the spiny lobster Panulirus interruptus." A cellular clotting protein called coagulogen with M, 70 kDa is found in the hemo- cytes of the sand crab Ovalipes bipustulatus. The coagulogen is induced to clot in the presence of human thrombin, human factor XII (TGase), and Ca**” ‘Thus, both cellular and humoral clotting proteins in ccrustacea are associated with the clotting reaction of hemolymph. In this study, we isolated HDL and VHDL from the hemolymph of sand crayfish by a density gradient ultracentrifugation. Sand crayfish had large amounts of VHDL, but not HDL, in their hemolymph, unlike other crustacea reported to date for lipoproteins. The N-termi- nal amino acid sequence of the VHDL was similar to those of clotting proteins from spiny lobster and crayfish. The VHDL seemed to serve as a clotting pro- tein in their hemolymph. This was also confirmed by cubating the VHDL with TGase and Ca. Materials and Methods Animals. Live male (average body weight 136 g) and female (average body weight 188 g, average egg weight ‘To whom correspondence should be addressed: Masaharu Komatsu, Ph.D., Department af Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 838-1 Sakuragaoka, Kagoshima 890, Japan. (Fax: 089-265-9687; E-mail Komatsu @khosp3 kutm.kagoshima-u.ae,p)460 M, Kowxrsu and S. ANDo 14) sand crayfish, Ibacus ciliatus, were purchased from a local fish market. Isolation of hemolymph lipoproteins. Hemolymph was withdrawn by syringe from sinuses at the bases of walking legs. The hemolymph was placed in equal volumes of 0.15 NaCl-10 mm phosphate buffer (pH 7.4) containing 3 mM EDTA, 3 mv dithiothreitol, 1 mM leupeptin, 2mm chymostatin, 3m pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1,000 KIE of aprotinin (KIB is defined as an amount of aprotinin necessary to decrease kallikrein activity from 2 to 1 unit at pH 8.0 and room temperature in 2hr of incubation; Bayer Leverkusen, Germany), and 2mw__ diisopropyl Muorophosphate. Following centrifugation (8,000 rpm at 4°C for 15 min) to remove hemocytes, the hemo- lymph was brought to a density of 1.35 g/ml with solid KBr (20 ml final volume), overlaid with 20 ml of 33% KBr in 0.15 m NaCl ~3 mm EDTA (pH 7.4), and cen- trifuged at 35,000 rpm for 17 hr at 15°C in a 410 rotor using an International B/60 model ultracentrifuge (DAMON/ IBC). The floating HDL and/or vitellogenin (VTG) which is a female-specifc lipoprotein and precur- sor of egg yolk protein were collected and adjusted to density of 1.25 g/ml by addition of solid KBr in a final volume of 20 ml. The solution was then placed in a cen- trifuge tube, overlaid with 20 ml of 119 KBr in 0.15 m NaCl-3 msi EDTA (pH 7.4), and respun at 35,000 rpm for IT hr at 15°C. The VHDL fraction (4=1.27-1.29 ¢/ mi) was respun under the same conditions of the first ultracentrifugation and completely separated from hemocyanin. After centrifugation, 40 fractions were col- lected by pipette and the density was measured by refrac- tometry. Sodium dodecylsulfate-polyacrylamide gel electropho- resis (SDS-PAGE) of apolipoproteins. SDS-PAGE was done by the method of Laemmli.” Electrophoresis was done on a gradient gel (4.5-18% polyacrylamide) at 20 mA for 3 hr under reducing or non-reducing conditions. In the reducing conditions, lipoprotein samples con- tained 1% 2-mercaptoethanol and were heated to 95°C for Smin. Protein bands on gels were stained with 0.25% Coomassie Brilliant Blue R-250. The relative molecular mass of apolipoproteins was measured by comparison with simultaneously run proteins of known. molecular mass (HMW Kit E (SDS) and LMW Kit E from Pharmacia LKB Biotechnology). Lipid and protein assays of lipoproteins. ‘The lipid compositions of lipoproteins were estimated using com- mercially available enzymatic kits from Kyowa Medex for triacylglycerol (TG), PL, free (FC), and total cholesterol (TC). The amount of cholesterol ester (CE) was calculated using the following formula: CE=1.61 x (TC-FO), where 1.61 is the conversion fac tor of cholesterol to CE. Protein concentration was de- termined using Bio-Rad Protein Assay Kit with bovine serum albumin as a standard. N-terminal amino acid sequence of VHDL. N-termi- nal amino a sequencing was done by the method of Matsudaira.”” Purified VHDL was subjected to SDS- PAGE (4.5-18% polyacrylamide gradient gel) under reducing conditions, and transferred by electroblotting, to polyvinylidene difluoride membrane (Nihon Milli- pore, Tokyo, Japan) in 10ms@ CAPS transfer buffer (pH 11.0) containing 10% methanol at 85 mA for 3 hr at 4°C. The membrane corresponding to an apolipoprotein with M, 195 kDa was then cut into 35 mm strips, and sequenced by Applied Biosystems model 492 gas phase sequencer. Clotting assay of VHDL. Guinea pig liver TGase (EC 2.3.2.13) was purchased from Sigma. The lyophilized powder containing TGase (2U), dithiothreitol, and Tris ‘was dissolved in I ml of Milli Q water. The VHDL (10 img protein/ml) was dialyzed against 0.15 m NaCl-50 mM Tris-HCI (pH 8.0). To the VHDL (0.9 ml) were added samples of 50 ul of TGase and either 50 yl of 1 M CaCl, of 50 ul of 0.2 M EDTA (pH 8.0). After the tilted test tubes containing the samples were left for Shr at 20°C, they were slightly shaken to observe clot forma- tion Results and Discussion Hemolymph lipoprotein profiles Insufficient dilution, less than one-half, or low speed centrifugation (3,500 rpm) in collecting hemolymph from sand crayfish resulted in occationally the clotting of VHDL after ultracentrifugation. We could obtain the intact VHDL only by the procedure described in “Materials and Methods”. Figure | shows the lipoprotein profile in the male sand crayfish hemolymph using first density gradient ultracen- ttifugation with the 1.21-1.35 g/ml density range. The VHDL (d= 1.27-1.29 g/ml) with orange coloration was separated from the blue-colored bottom fraction con- taining hemocyanin. The bottom fraction contained a small amount of PL and TG, suggesting some contai nation of VHDL in this fraction, Although the protein concentration in each fraction was different between male and female, the lipoprotein profile of female was similar to that of male. The VHDL fraction separated from hemocyanin was pooled and respun for further purification. ‘The protein peak corresponding to HDL could not be detected after the first density gradient ultracentrifuga- tion as shown in Fig. 1, while pale orange coloration was observed at the top layer of the centrifuge tube. Then, the top layer was ultracentrifuged on a second density gradient with the 1.06-1.25 g/ml density range to confirm the presence of HDL (Fig. 2). A small amount of HDL (d=1.16 g/ml) was isolated from the male hemolymph, while a shoulder peak (d= 1.15 g/ml) corresponding to HDL was found in the female hemo- lymph along with a main peak of VTG (d= 1.19 g/m)) The presence of VTG in the female hemolymph was closely associated with the eggs attached to the pleopods under the abdomen.” The HDL level in the female hemolymph was as little as that in the male. Both HDL. and VTG isolated by the second ultracentrifugationVHDL with Clotting Ability in Sand Crayfish 461 20 71.96 134 192 1.0 128 126 Density (g/m!) 1.24 Protein concentration (mg/m!) Lipid concentration (mami) 1.22 Fraction number Fig. L._Distribution of Density (+), Protein (©), Triacylglycerol (C2), and Phospholipid (@) in the Fractions from the Hemolympi of Male Sand Crayfish after Density Gradient Ultacentrifugation. ‘The hemolymph was applied to density gradient ultraceneiusa sion (1.21-1.38 g/ml density range). Tubes were fractionated in I ri fractions from thetop layer. The density was measured by refrac tometey if “| HoH bs “| I: | Fig. 2. Density Gradient Ultacentrifugation of High-density Lipoprotein Fractions from Male (A) and Female (B) Sand Crayfish ‘Tubes were fractionated in Ll fractions from the top layer after ‘ultracentrifugation (1,06-1.25 g/ml density range), The density was reasured by refractomenry. ©, Protein; ©, tiacylalycerol phospholipid. showed pale orange coloration. ‘We had previously isolated not only HDL but VHDL from the hemolymph of some crustacea."" They con- tained HDL and VHDL in the hemolymph ranging in Table 1. Chemical Composition of Hemolymph Lipoproteins of sand Crayfish - Concentration (mai hemolymph) HDL VIG VHDL Male Female Female Male Female Protein 0.029 O08 0388 21.380 4.990 “riacyetyeerol 0.005 0.033 0.187 0.066, Phospholipid 0.09 0.168 Lom? 0.270, Free cholesterol 0.001 0.006 0.048 0.006, Cholesterol eter 0.0008 0.001 0.002 0.045 0.008 Ratio (ProtincLipid) 1087 10.87 120.60 10.06 1-007 Density (s/s) 1618 Lay 1.29 1.27 concentrations from 0.2 to 3.3 mg/ml and 0.3 to 6.5 mg/ml, respectively. The lipid concentration of VHDL was much lower than that of HDL reflecting the differ- ence in their densities. This suggested that HDL, but not VHDL, was the main carrier of lipid in crustacea. The lipoprotein profile of sand crayfish was apparently differ- ent from those of other crustacea (Table I). The VHDL was the most abundant lipoprotein in both male and fe- male sand crayfish, although the male had much more VHDL in the hemolymph than the female. Calculating the lipid concentrations in the lipoprotein fractions from male sand crayfish, most lipids were distributed in the VHDL, but not HDL. A similar distribution of lipid in the lipoprotein fractions was also found in female sand crayfish, except for the VTG which contained one- third of the hemolymph lipids. Phospholipid was the main lipid component in all lipoproteins isolated from the hemolymph of sand crayfish. Thus, sand crayfish had a unique lipoprotein profile in the hemolymph, different from other crustacea reported to date." Apolipoprotein features Figure 3 shows apolipoprotein components of VHDL, VTG, and HDL isolated from the hemolymph of sand crayfish. The VHDL had some dominating subu- nits with more than M, 400 kDa and a minor component with M, 175 kDa under non-reducing conditions. Reduc- ing conditions of VHDL resulted in the appearance of a main subunit with M, 195kDa, indicating this apolipoprotein consisted of dimers by disulfide bonds. The behavior of main apolipoprotein of VHDL in SDS- PAGE was exactly the same as that of clotting protein with M, 210kKDa in crayfish." The main apolipoprotein with M, 195 kDa of VHDL was compara- ble to the clotting protein in crayfish. The VTG isolated from the hemolymph of female sand crayfish consisted of two apolipoproteins with M, 20S kDa and M, 85kDa. The HDL from male sand crayfish had one apolipoprotein with M, 120 kDa, while some minor components with M, 205 kDa and M, 85 kDa as well as a main apolipoprotein were observed from the female HDL. This suggested the HDL isolated from the female was contaminated by the VTG. The crustacean HDL described so far had an apolipoprotein with molecular mass ranging from 100 to 120 kDa.*"™ ‘The apolipoprotein of HDL seemed to be very well con-462 IM. Komarsu and 8, ANDo A BcoODE >400kDae— 205kDa 175kDar--195kDar em 120 kDa od a5kDa > Fig. 3. Sodium Dodecylsulfate (SDS)-Polyacrylamide Gel Elec Trophoregrams of Very-high-density Lipoprotein (VHDL), Vite. logenin (VIG), and High-density Lipoprotein (HDL). 'SDS-polyacrslamide gel eletrophoress was done under reducing GC, D, E) or non-reducing condition (A) using 45-18% gradient sel and 10g lipoproteins were put on the gel. A and B, VHDL from male sand crayfish; C, VIG from female sand erafish; D and E, HDL from female and male sand crayfish, respectively served throughout crustacea. Clotting ability of VHDL Hall e¢ al. recently showed that the hemolymph lipoproteins from crayfish had dual functions associated with lipid carriers and defense reactions."°" The HDL. and VHDL isolated from the hemolymph of crayfish were identical to f-1,3-glucan binding protein and clot- ting protein, respectively, which had been characterized regardless of hemolymph lipoproteins. The VHDL isolated from the hemolymph of sand crayfish seemed to be similar to the clotting protein in crayfish as described previously. The clotting protein is a substrate for TGase by which covalent crosslinks be- ‘tween specific lysine and glutamine residues are formed on the substrate, resulting in the clotting of whole hemo- lymph.“ We observed the clotting ability of VHDL. isolated from sand crayfish using guinea pig liver TGase. ‘The VHDL could be converted into a clot if incubated with TGase and Ca** (Fig. 4). The presence of Ca** was necessary for the clotting of VHDL and EDTA inhibit- ed the clotting reaction of VHDL completely. ‘The N-terminal amino acid sequence of clotting pro- teins with M, 220 k and 210 kDa has so far been report- ed for spiny lobster” and crayfish," respectively. Although the clotting proteins of spiny lobster and crayfish were first isolated from their hemolymph without knowledge of lipoprotein, the latter has proved to be identical to VHDL as described previously. We analyzed the N-terminal amino acid sequence of VHDL. isolated from the male sand crayfish and compared it with those of spiny lobster and crayfish clotting proteins (Fig. 5). The N-terminal of sand crayfish VHDL was identical with 12 amino acids of the clotting proteins from both crustacea among 17 amino acids. These results shown in Fig, 4 and 5 strongly suggested that the VHDL isolated from sand crayfish contained a clotting protein and that the clotting protein of spiny lobster was identical to VHDL. The clotting proteins called fibrinogen and coagulo- gen have been investigated in detail in humans” and xiphosuran horseshoe crabs™” which are different from ABc OD 4. Clotting Ability of Very-highlensity Lipoprotein (VHDL) Isolated fom Male Sand Crayfish To the VHDL (9 me as protein) were added samples of 50 (0.1 of eunies ansglutaminase (TGase) and ether 50, of 1a CaCl, (A) oF $0 of 0.2 EDTA (B). The tilted test tubes con- taining samples were left for $ heat 20°C and slightly shaken to ob- serve elot formation. C, No TGase in A; D, No VHDL in A. Sand crayfish: 2 QP OLEYOYR YN ‘Spiny lobster : 1 @ P Craytish «ss Fig. 8. Noemi Lipoprotein Isolated from Male Sand Crayfish ‘Comparison of the N-terminal amino ack sequences of clotting proteins from spay'Tobster® and crayfish. The shadowed indicate identical amino acids crustacea, respectively. The clotting process is elaborat- ed in these species and various types of serine proteases are involved in this reaction. In the horseshoe crab, coagulogen is converted to coagulin gel by a clotting en- zyme that induces limited proteolysis of the precursor. ‘The clotting enzyme in horseshoe crabs is similar to a- thrombin, which is associated with the conversion of fibrinogen to fibrin in humans. The fibrin finally forms a stable gel via the action of TGase.”” Since the VHDL of sand crayfish was induced to clot only by the presence of TGase and Ca** (Fig. 4), the clotting process in crustacea such as sand crayfish, crayfish, and spiny lob- stet was different from that in horseshoe crabs." Considering large amounts of VHDL, but not HDL, in the hemolymph of sand crayfish, the VHDL is a cru cial component as not only a lipid carrier but for the clot- ting process. Acknowledgment We are indebted to Dr. S. Hayashi for ment. References 1D) A.M, Gott, Je.,H. J. Pownall, and R, J. Havel, in “Methods in Enzymology", Vol 128, ed. by J.P. Segrest and J.J. 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