J. Biochem. 101, 545-551 (1987) Purification of a Lectin from the Hemolymph of Chinese Oak Silk Moth (Antheraea pernyi) Pupae1 Xian-Ming QU,2 Chun-Fa ZHANG,3 Hiroto KOMANO, and Shunji NATORI Faculty of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, November 7, 1986 A lectin with affinity to galactose was purified to homogeneity from the hemolymph of diapausing pupae of the Chinese oak silk moth, Antheraea pernyi. The molecular mass of this lectin was 380,000 and it formed an oligomeric structure of a subunit with a molecular mass of 38,000. The hemagglutinating activity in the hemolymph was found to increase with time after immunization with E. coli. Studies with antibody against the purified lectin showed that increase in the hemagglutinating activity was due to the same lectin, suggesting that the amount of the lectin increased in response to intrusion of foreign substances. The function of this lectin in the defence mechanism is discussed. The hemolymphs of many invertebrates are known have no definite humoral immune system like that to contain agglutinin (1-7). Most of these agglu in vertebrates, one of the biological roles of inver tinins are proteins binding carbohydrates, so they tebrate lectins has been suggested to be in the can be defined as invertebrate lectins. These lec defence mechanism (8-11). However, no conclu tins differ greatly in molecular masses, subunit sive evidence to support this idea has yet been structures, hapten sugars, and ionic requirements obtained. (7). Probably, these lectins participate in many Previously, we purified a lectin from the aspects of fundamental biological events, such as hemolymph of Sarcophaga peregrina (flesh-fly) development, differentiation, recognition of self larvae (12). This Sarcophaga lectin is peculiar in and non-self, and so forth. Since invertebrates the following two points: 1) Normal larvae do not contain this lectin, but it is promptly induced in 1 This work was supported in part by a Grant-in-Aid the hemolymph on injury of the body wall (12, 13). for Scientific Research from the Ministry of Education, Namely, this lectin is induced under conditions Science and Culture of Japan. when the defence system of this insect is supposed 2 Present address: Shanghai Institute of Biochemistry , to be enhanced. Consistent with this possibility, Academica Sinica, 320 Yue-Yang Road, Shanghai, we showed that this lectin participates in the elim China. ination of sheep red blood cells introduced into the 3 On leave of absence from the Silkworm Research In abdominal cavity of larvae (14). 2) This lectin is stitute, Fengcheng, Liaoning Province, China. Abbreviations: SDS, sodium dodecyl sulfate; kDa, synthesized not only on injury of the body wall of larvae, but also in the early embryonic stage and Vol. 101, No. 3, 1987 545 546 X.-M. QU, C.-F. ZHANG, H . KOMANO, and S. NATORI in the early pupal stage in normal development of buffer, pH 6.4, containing 0.13 M NaCl, 5 mM this insect (15). Thus, it may have functions in KCl, and 1 mM CaCl2 by extensive washing. The both the defence mechanism and development. If final preparation was stored at 4•Ž. such lectins are essential for development, they are Molecular Weight Determination by Gel Filtra probably present in all holometabolous insects. tion•\A column of Toyopearl HW-55 (Toyo Soda, We found a similar lectin in the hemolymph Japan, 1 •~ 80 cm) was equilibrated with 10 mM of pupae of the Chinese oak silk moth, Antheraea phosphate buffer, pH 7.4, containing 130 mM NaCl pernyi. This paper describes the purification and , 3 mM KCl, and 0.2 M glucose. The pro some characteristics of this Antheraea lectin. tein sample (180 ƒÊg/0.5 ml) was applied to the column, and chromatography was carried out at MATERIALS AND METHODS room temperature at a flow rate of 2 ml/h. Frac tions of 1 ml were collected and assayed for hemag Animals and Collection of Hemolymph•\Di glutinating activity. The column was calibrated apausing pupae of the Chinese oak silk moth, A. with bovine serum albumin (68,000), aldolase pernyi, were supplied from the Liaoning Province (158,000), ferritin (450,000), and thyroglobulin Silkworm Research Institute, China. Pupae were (670,000). stored at 4•Ž. Under these conditions, pupae Polyacrylamide Gel Electrophoresis•\Electro could be kept for at least 6 months without devel phoresis on polyacrylamide SDS-slab gel was car opment. For collection of hemolymph, the poste ried out by the method of Laemmli (16). Proteins rior tip of the pupae was cut off with fine scissors were denatured by heating them in 1% SDS solu and the hemolymph that exuded was collected in tion containing 2% 2-mercaptoethanol for 20 min an ice-cooled test tube containing a few crystals at 75•Ž. The stacking gel contained 3% acryl of phenylthiourea to inhibit the activity of phenol amide and the separating gel contained 12.5% oxidase. Hemocytes were removed by centrifuga acrylamide. After electrophoresis, the gel was tion for 30 min at 10,000 •~ g, and the resulting stained by the method of Fairbanks et al. (17). clear supernatant was stored at -20•Ž. Usually, For estimation of the molecular mass, the gel was 200 ml of hemolymph was collected from 100 calibrated with bovine serum albumin (69,000), pupae. ovalbumin (45,000), and chymotrypsinogen Assay of Hemagglutinating Activity•\Hemag (25,000). glutinating activity was assayed essentially as de Antibody against Antheraea Lectin•\Antibody scribed before (12), with commercially available against Antheraea lectin was raised by injecting 500 rabbit red blood cells as an indicator. Unagglu ƒÊg of purified lectin mixed with complete Freund's tinated cells formed a clear dot, whereas agglu adjuvant into a male albino rabbit. Injections tinated cells formed a diffuse mat on the bottom were made into the footpads and also to five to of wells of microtiter V-plates. The end point of six sites in the back. A booster injection of the transition from a diffuse mat to a dot was distinct same amount of the lectin mixed with incompleted on dilution of the hemolymph. Hemagglutinating Freund's adjuvant was given two months later, activity was defined as the reciprocal of the maxi and the animal was bled one week after the booster mum dilution of the test sample causing hemag injection. The reactivity of the antiserum was glutination (titer). determined by the double gel diffusion method of Acid-Treated Sepharose 6B•\Acid-treated Se Ouchterlony (18). Immunoglobulin G (IgG) was pharose 6B was prepared as follows: About 150 purified from the serum by the method of McCauley ml of Sepharose 6B (Pharmacia) was suspended in and Racker (19). Protein was determined by the 500 ml of 0.2 N HCl and washed well with distilled method of Lowry et al. (20). water. The resulting paste was suspended in 240 ml of 0.2 N HCI and kept at 53•Ž for 2 h with RESULTS gentle shaking. The acid-treated Sepharose 6B thus obtained was washed extensively with distilled Hemagglutinating Activity of Hemolymph of water, transferred to a column (4 •~ 27 cm), and Antheraea Pupae•\Significant hemagglutinating ac equilibrated with 10 mM CH3COOH/CH3CONH2 tivity was detected in the hemolymph of pupae of J. Biochem . PURIFICATION OF Antheraea LECTIN 547 TABLE I. Effects of sugars on hemagglutinating activity. Fig. 1. Enhancement of the hemagglutinating activity in the hemolymph of Antheraea pupae on injection of E. coll. A suspension of 105 E. coli in a volume of 50 ƒÊl was injected into each pupa cells at time 0. Pupae were kept at 4•Ž. As controls, normal larvae were kept under the same conditions. Hemolymph samples a 20-Fold diluted hemolymph was used . were collected at the indicated times and their hemag glutinating activity was measured. Each point repre sents the average value for four pupae. •œ immunized sugar specificity or to multiple lectins each with a pupae; •›, normal pupae. different sugar specificity. To clarify this point, we tried to purify this lectin(s). the Chinese oak silk moth, A. 1pernvi, when assayed Purification of Antheraea Lectin-Since the with rabbit red blood cells as an indicator. This hemagglutinating activity in the hemolymph was activity increased markedly with time after injec inhibited by n-galactose, the lectin could be puri tion of a suspension of E. coli into the pupae (105 fied by affinity chromatography on galactose. cells/animal), as shown in Fig. 1. Similar increase About 235 ml of hemolymph collected from 100 in the production of antibacterial proteins termed pupae was heated at 70•Ž for 15 min to reduce cecropins was observed in the pupae after the same its viscosity, and then cooled and centrifuged for treatment (21). Thus, this hemagglutinin may well 20 min at 10,000 •~ g to remove denatured proteins. be a sort of defence protein like Sarcophaga lectin The resulting clear supernatant was applied to a found in the hemolymph of S. peregrina larvae column of acid-treated Sepharose 6B (4x 27 cm) when their body wall is injured with a hypodermic equilibrated with 10 mM acetate buffer. Under needle, and it may function in the elimination of these conditions most proteins did not bind to the foreign substances introduced into the abdominal column and were recovered in the flow-through cavity. fraction. But this fraction did not contain ap This hemagglutinating activity was found to preciable hemagglutinating activity. The bound be inhibited by a-methyl-D-glucoside and D-galac material was eluted with 0.2 M galactose. Each tose at concentrations of 5 and 25 mM, respec fraction of eluate was dialyzed extensively against tively, as shown in Table I.