Fisheries Science 62(1), 138-141 (1996)

Characterization of the Lectin from the Skin Mucus of the Kingklip capensis

Michitoshi Toda, Rina Goto-Nance, Koji Muramoto,•õ and Hisao Kamiya

Department of Marine Biochemistry, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan

(Received June 26, 1995)

A mitogenic lectin in the skin mucus of the kingklip Genypterus capensis was purified by a combi nation of affinity adsorption on the glutaraldehyde-fixed rabbit erythrocyte ghosts and chromatofocus ing on PBE 94 gels. The lectin was a glycoprotein having a molecular weight of 28-34kDa which was composed with two homogenous subunit (13.7kDa). The isoelectric point was 6.45. Kingklip lectin ag glutinated rabbit and horse eryttirocytes but not human ABO erythrocytes. The hemagglutinating activi ty was calcium ion-dependent and was inhibited effectively by asialo-mucin Type I and also by simple su gars such as N-acetyl-D-glucosamine. The amino-terminal sequence of the kingklip lectin was identified as Ser-Met-Cys-Asn-Cys-Gly-Trp-Ser-Gln-Phe-Ala-His-Leu-Ala-Tyr-Leu-Leu-Arg-Ser-Lys-Ala- . Key words: Genypterus capensis, kingklip, skin mucus, lectin, mitogenic

Skin mucus of fish serves as a lubricant in locomotion

machines and has mechanical protective functions. Mucus Materials and Methods is a complex mixture of materials; the major component is

mucin, composed mostly of glycoproteins, and other com Preparation of Skin Mucus Extract ponents including immunoglobulins, complement, C-reac Frozen specimens of the kingklip Genypterus capensis tive protein, lysozyme, and hemolysins.1) In addition to caught off New Zealand were provided by Maruha Corpo these components, natural lectins or carbohyrate-binding ration Central Research Institute. Skin mucus was scraped proteins have been reported to occur in the skin mucus.2-8) off from the fish and was extracted twice with an equal As to the physiological roles of these skin mucus lectins, a volume of 50 mm Tris-HCl buffer, pH 7 .4 (THB), and cen function in the defense mechanism is proposed: The skin trifuged at 15,000 x g for 20 min at 4•Ž . The supernatant mucus lectins function as inhibitors against colonization combined was dialyzed against 50 mm THB for 48 h. The or invasion of potential pathogenic microorganisms. In retentate was kept frozen until use. fact, lectins in the fish species such as windowpane flound er 21and conger ) have been reported to agglutinate ma Agglutination Test rine microorganisms, but no growth inhibition of marine Serial 2-fold dilutions of the mucus extract (50ƒÊl) were

microorganisms by these lectins has been recognized. made in multiwell microtiter plates using 0 .15 M NaCl with These observations make the functions of fish skin mucus or without 10 mm CaCl2 as a diluent. Estimation of aggluti

lectins indistinct. On the other hand, the recent accumula nation activity against horse , rabbit, and human ABO tion of data on lectin sequences enables us to classi erythrocytes was carried out usmg a 4% erythrocytes sus fy the lectins into several types and postulate the physiolog pension (50ƒÊl). After allowing the microtiter plates to ical functions to some extent by comparing the sequences stand at 6, 20, or 37•Ž for 1 h, hemagglutination titer was to other proteins.9-11) In the case of fish skin mucus, only recorded as the reciprocal of the maximum dilution giving the sequence of conger mucus has been reported.12) positive agglutination. A heat stability test was performed Therefore, more information of chemical as well as biolog by incubating the diluted mucus extract at 40 , 60 and 80•Ž f ical properties is inevitable to know the status of these fish or 5 min. After immediate cooling , the hemagglutination skin mucus lectins. activity was determined as described above . The effect of In our survey on fish mucus lectins, we found that the pH on the agglutinating activity was examined by adjust skin mucus of the kingklip Genypterus capensis contained ing the pH values of the mucus extract to 2 , 4, 6, 8, 10, and 12 a new lectin which showed mitogenic activity for T lympho with 0.1ml NaOH or HCl . After keeping the sample at cytes but not for B lymphocytes.13) We have isolated this 6•Ž overnight, the residual activity was estimated at pH lectin in an electrophoretically pure form and found this 7.4. The calcium ion dependency of the hemagglutinating lectin resembles to the C-type lectins.91 This paper deals activity was examined as follows . One ml of the mucus ex with the isolation and characterization of the lectin from tract was dialyzed against 25 mM EDTA in 0 .2 M THB for h24 G. capensis skin mucus. , and then against 0.85% NaCl for 24 h . An aliquot was tested for hemagglutinating activity in the presence of

•õ Present address: Faculty of Agriculture, Tohoku University, Sendai 981, Japan. Lectin in the Kingklip Skin Mucus 139

CaC12 and in 0.15M NaCl. Preparation of Glutaraldehyde-Fixed Ghost as an Affinity Crossed absorption test was carried out as follows. The Adsorent skin mucus extract was mixed with a half-volume of pack Rabbit erythrocytes were separated from 200 ml of ed rabbit or horse erythrocytes and incubated for 2 h at blood by centrifugation at 1000 x g for 20 min and washed room temperature. After centrifugation, the supernatant thrice with 50 mm THB containing 0.15M NaCl. To the was tested against the same erythrocyte type used for ab washed rabbit erythrocytes were added 20 volumes of 10 sorption and against the other erythrocyte type. mm THB to occur hemolysis and stirred gently for 15 min The agglutinating activity against marine bacteria, Pseu at 6•Ž. The hemoglobin-free ghosts were centrifuged at domonus fluorescens and Vibrio anguillarum was also 15,000 x g for 40 min at 6•Ž, washed 5 times with 10 mm examined using filtered sea water. Agglutination was ob THB, and fixed with 1% glutar aldehyde at a room tem served under a microscope. perature for 5 h. The reaction was terminated by adding 0.1 volume of 1 M L-lysine. The glutaraldehyde-fixed Hemagglutination Inhibition Test ghosts were washed 5 times with 50 mm THB containing Inhibition of hmagglutination against rabbit erythro 0.15 M NaCl, suspended in 50 mm THB containing 0.15 M cytes by sugars and glycoproteins was done using the NaCl and 20 mm CaCl2, and stored at 6•Ž. mucus extract. The soluions were allowed to react with various concentrations of carbohydrate for 1 h, and then Isolation of G. capensis Skin Mucus Lectin the mixture was tested for its hemagglutinating activity. The mucus extract (20 ml, 600 mg protein) was mixed The following sugars (200 mm) and glycoproteins (0.25%) with an equal volume of a suspension of glutaraldehyde were tested: L-fucose, D-ribose, L-arabinose, L-rhamnose, fixed ghosts at a room temperature for 4 h in the presence D-galactose, D-mannose, D-xylose, D-glucose, lactose, gen of 10 mm CaCl2. After the incubation, the mixture was cen tiobiose, D-mannosamine, D-glucosamine, D-galactosa trifuged at 15,000 x g for 15 min. The supernatant showed mine, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, no hemagglutinating activity. The precipitate was then N-acetyl-D-mannosamine, fetuin, mucin Type I from bo washed 5 times with 50 mm THB containing 0.15 M NaCl vine submaxillary gland (BSM), and mucin Type ‡U from and 10 mm CaCl2. The precipitate was suspended in 7 ml

porcine stomach (PSM), asialo-BSM, and asialo-PSM. of 0.1 M THB containing 10 mm EDTA at 6•Ž overnight and centrifuged at 15,000 x g for 15 min. The ghosts were Analytical Methods precipitated, while the lectin was released in the super Protein concentrations were determined by the method natant. The supernatant was dialyzed against 10 volumes of Bradford14) with bovine serum albumin as a standard. of 50 mm THB containing 0.15 M NaCl and 10 mm CaCl2 Sodium dodecyl sulfate-polyacrylamide gel electrophore for 24 h. For further purification, chromatofocusing using sis (SDS-PAGE) was performed in a 12% or 15% poly PBE 94 gel and Polybuffer 74 (Pharmacia, Uppsala) was acrylamide gel. The gel was stamed with 0.1% Coomassie adopted according to the instruction manual by the brilliant blue R-250. Electrofocusing on a 5% poly manufacturer. Briefly, the partially purified lectin (7 ml, acrylamide gel was carried out at 200 V for 4 h. Half of the 3.5 mg protein) was dialyzed against 25 mm immidazol

gel was used for staining with Coomassie brilliant blue. - HCl buffer, pH 7.4 for 24 h, applied toacolumn (1.1 x 24 The other was cut into 5 mm slices and each slice was used cm) of PBE 94, and developed with a diluted Polybuffer for measurement of pH and hemagglutinating activity af 74, pH 4.0. Fractions of 1.5 ml were collected and ana ter extraction with 0.5 ml of 0.15 M NaCl. The molecular lyzed for the absorption at 280 nm, pH values, and hemag weight of the purified lectin was determined by fast protein glutinating activity against rabbit erythrocytes. To the ac liquid chromatography (FPLC) on a Superose 12 HR10/ tive fractions that combined was added solid ammonium 30 column. sulfate to give a final concentration of 80% saturation. Analysis of amino acid composition was carried out The mixture was centrifuged at 15,000 x g and the using the purified lectin which was electroblotted onto a precipitate obtained was dialyzed against 50 mm THB con polyvinylidene difluoride (PVDF) membrane after SDS taining 0.15 M NaCl and 10 mm CaCl2. - polyacrylanilde gel electrophoresis.15) Briefly, the samples were hydrolyzed in 6N HCl containing 1 % phenol at Results 110•Ž for 22 h and derivatized with DABS-Cl.16) Analysis was performed on a column of Capcell Pak C18 AG120A Hemagglutination Activity of G. capensis Skin Mucus

(5 mm, 4.6 x 150 mm) maintained at 40•Ž. The elution Extract solvent system was a 25 mm sodium acetate buffer (pH The skin mucus extract agglutinated rabbit and horse 6.5) containing 4% dimetylformainide and acetonitrile. erythrocytes but not human ABO erythrocytes. The For analysis of reducing sugar composition, 5ƒÊg of the hemagglutinating activity by the lectin varied markedly ac sample was hydrolyzed in 2 M trifluoroacetic acid. The cording to the incubation temperatures, and the highest DABS-hydrazide derivatives were separated by reversed titer (512) was observed at 37•Ž to rabbit erythrocytes. At phase HPLC.17) 20•Ž, the titer of the extract decreased to 128, and the Amino-terminal sequence of the electroblotted lectin lowest titer (2) was observed at 6•Ž. The lectin was heat was analyzed in a Shimadzu gas-phase sequencer PSQ-1. labile and the initial activity was lost on heating at 60•Ž SWISS-PROT protein-sequence-database, Rel.30, was for 5 min. The activity remained unchanged over relatively used to obtain amino-acid sequence information. narrow range of pH values between 6 and 8. It was inacti vated at pH 2 and pH 12. The mucus extract lost its activi ty after dialysis against EDTA. The addition of calcium 140 Toda et at. ions restored the initial hemagglutinating activity indicat ing the dependency of the activity on the presence of calci um ions. The mucus extract did not agglutinate marine bacteria tested. The extract agglutinated both rabbit and horse erythro cytes. It was important to know that two different lectins were present in the extract or a single type of the lectin reacted with both. In the crossed absorption test, the pack ed rabbit erythrocytes absorbed the hemagglutinating activity to horse erythrocytes and vice versa. These results allowed us to use rabbit erythrocytes as target cells in the following experiments. In hemagglutination inhibition test by simple sugars and Fig. 1. Purification of G. capensis skin mucus lectin by chromatofocus glycoproteins, asialo-BSM was found to be the most po ing. tent inhibitor as shown in Table 1. Simple sugars such as Affinity purified lectin was applied to a column (1.1 x 24 cm) of N-acetyl-D-glucosamine, D-mannose and L-fucose were PBE 94, and the column was developed with 300 ml of a diluted also inhibitors but required much higher concentrations to Polybuffer 74, pH 4.0. Fractions of 1.5 ml were collected and ana inhibit the hemagglutinating activity compared to asialo lyzed for the absorption at 280 nm (•œ-•œ), pH values (•›...•›), and hemagglutinating activity against rabbit erythrocytes. Active BSM. On the other hand, glycoproteins except asialo-BSM fractions (indicated by a horizontal bar) were combined and concen did not exhibit any inhibitory activity. trated.

Purification and Characterization of G. capensis Skin Mu cus Lectin G. capensis lectin was purified effectively using rabbit glutaraldehyde-fixed ghosts as an affinity adsorbent. Suc ceeding chromatofocusing on PBE 94 gels gave a single protein peak showing the hemagglutinating activity against rabbit erythrocytes (Fig. 1). In SDS-PAGE, the purified lectin gave a single protein band with or without a reducing agent (Fig. 2). From the relative mobility of the purified lectins in a 12% and 15% gel, the molecular weight was estimated to be 13.7KDa under reduced condi tions and 28kDa without a reducing agent. On the other hand, the molecular weight of the intact lectin was esti mated to be 34kDa by FPLC on a Superose 12 column. These indicated that the lectin molecule is composed of two covalently bonded subunits. The mucus extract (600 mg protein) yielded 2.24 mg pro tein of the purified lectin. The overall yield relative to the initial total activity was 40%. The purification fold of the Fig. 2. SDS-polyacrylamide gel electrophoresis of G. capensis skin mu final preparation was about 480. cus lectins on a 12% gel. The lectin was a glycoprotein in nature. The neutral (A) Standard proteins, (B) extract of skin mucus, (C) affinity sugar composition was 0.5% D-glucose, 0.4% D-mannose, purified lectin, (D) the purified lectin, and (E) the purified lectin. (A) and 0.6% D-xylose. The hemagglutination by the purified and (E) under reducing conditions; (B), (C), and (D) without a reduc ing agent. Standard proteins used: phosphorylase b (94kDa) , bovine lectin was inhibited by the same kinds of sugars and serum albumin (64kDa), ovalbumin (43kDa), carbonic anhydrase glycoproteins as observed for the mucus extract. In amino (30kDa), soybean trypsin inhibitor (20.1kDa), ƒ¿-lactalbumin (14.4 kDa). Table 1. Hemagglutination Inhibition of Genypterus capensis lec tin by Sugars and Glycoproteins

Table 2. Amino acid composition of Genypterus capensis skin mucus lectin

* The concentration of sugars and glycoproteins required for the complete inhibi tion of hemagglutination by the mucus extract (titer 256). Lectin in the Kingklip Skin Mucus 141 acid analysis of the electroblotted lectin to a PVDF mem lectin in the defense mechanism. The presence of lympho brane, Asx, Phe, and His were the major constituents as cytes or epithelial cells, which are responsible for the pro listed in Table 2. A relatively large amount of Glx, Ser, tection, under or in the epidermis of the fish receiving Ala, Leu, Lys, and Gly was recognized. Cys (half) was also stress or injury was observed (Y. Suzuki, person. comm.). detected in the purified lectin. The electric point of the It is of interest to know from the standpoint of physiology lectin was determined to be pH 6.45 by isoelectrofocusing. of lectins whether the mitogenic lectins in the skin mucus The amino-terminal sequence was identified as Ser-Met of the Ophidiidae stimulate any type of their own cells to Cys-Asn-Cys-Gly-Trp-Ser-Gln-Phe-Ala-His-Leu-Ala-Tyr grow. Leu-Leu-Arg-Ser-Lys-Ala-. Acknowledgments The authors express their sincere thank to Dr. S. Discussion Kimura, Director of Maruha Corporation Central Research Institute, for supplying us the specimens for this study. The lectin in the skin mucus of G. capensis was effective ly purified by a combination of affinity adsorption on References glutaraldehyde-fixed ghosts and chromatofocusing. G. 1) G. M. Ingram: Substances involved in the natural resistance of fish capensis skin mucus lectin was a glycoprotein having a to infection-A review. J. Fish. Biol., 16, 23-60 (1980). molecular weight of 28kDa. It split into a subunit show 2) H. Kamiya and Y. Shimizu: Marine biopolymers with cell specifici ing a molecular weight of 13.7kDa under a reduced condi ty II. Purification and characterization of agglutinins from mucus tion. In the amino acid sequence analysis of the amino-ter of windowpane flounder Lophopsetta maculata. Biochim. Biophys. minal region, we obtained a sequence with 21 amino acid Acta, 22, 171-178 (1980). residues. These results indicate that G. capensis lectin is 3) Y. Oda, S. Ichida, T. Mimura, K. Maeda, K. Tsujikawa, and S. composed of two identical subunits which are held Aonuma: Purification and characterization of fish lectin from the external mucus of Ophidiidae, Genypterus blacodes. J. Pharm. together by disulfide bond(s). G. capensis lectin resembles Dyn., 7, 614-623 (1984). to the lectin in the skin mucus of the ling G. blacodes, 4) Y. Suzuki and T. Kaneko: Demonstration of the mucus hemaggluti another member of the Ophidiidae, both in chemical struc nin in the club cells of eel skin. Dev. Comp. Immunol., 10, 509-518 ture and biological activity.3) The ling lectin (32kDa) con (1986). sisted of four covalently bonded subunits (8KDa) and 5) H. Kamiya, K. Muramoto, and R. Goto: Purification and proper showed a mitogenic activity against mice T lymphocytes.18) ties of agglutinins from conger eel, Conger myriaster (Brevoort), skin mucus. Dev. Comp. Immunol., 12, 309-31 (1988). However, the differences in the hemagglutinating activity 6) K. Shiomi, H. Uematsu, H. Ito, H. Yamanaka, and T. Kikuchi: and the sugar-binding specificity were recognized between Purification and characterization of a galactose-binding lectin from G. blacodes and G. capensis lectins. G. capensis lectin ag the skin mucus of the conger eel Conger myriaster. Comp. glutinated horse and rabbit erythrocytes but not any type Biochim. Physiol., 92B, 25-261 (1989). of human erythrocytes while G. blacodes lectin agglutinat 7) K. Shiomi, H. Uematsu, H. Ito, H. Yamanaka, and T. Kikuchi: ed these types of erythrocytes non-specifically. The differ Purification and properties of a lectin in the skin mucus of the ence was significant in the sugar-binding specificity against Repomucenus richardsonii. Nippon Suisan Gakkaishi, 56, 119-123 (1990). glycoproteins. The hemagglutination by the mucus xtract 8) R. Goto-Nance, Y. Watanabe, H. Kamiya, and H. Ida: Characteri as well as the purified G. capensis lectin was inhibited by zation of lectins from the skin mucus of the loach Misgurnus anguil asialo-BSM but not by BSM, PSM, and fetuin which have licaudatus. Fisheries Sci., 61, 137-140 (1995). been reported to be potent inhibitors for G. blacodes 9) K. Drickamer: Two distinct classes of carbohydrate-recognition lectin.3) domains in animal lectins. J. Biol. Chem., 263, 9557-9560 (1988). Animal lectins from various origins are categorized into 10) K. Drickamer and M. E. Tayler: Biology of animal lectins. Annu. Rev. Biol., 9, 237-264 (1993). two major groups, C-type and S-type lectin families, ac 11) A. J. Day: The C-type carbohydrate recognition domain (CRD) su cording to the sequence homology, especially in the carbo perfamily. Biochem. Soc. Transact., 22, 83-88 (1994). hydrate recognition domains, and also to calcium ion de 12) K. Muramoto and H. Kamiya: The amino-acid sequence of a lectin pendency of the activity.9) However, it is not clear whether from conger eel, Conger myriaster, skin mucus. Biochim. Biophys. the fish mucus lectins are classified or related to which type Acta, 1116, 129-136 (1992). of the lectin families to date. We have recently determined 13) H. Kamiya, K. Muramoto, R. Goto, and M. Yamazaki: Lectins in the complete sequence of congerin, the conger eel skin mu fish ucus. JADCI Meeting 1991, Dev. Comp. Immunol., 16, p.V, B3, 1992. cus lectin.12) Congerin is devoid of cysteine residue in the 14) M. M. Bradford: A rapid and sensitive method for the quantitation molecule and proved to be a member of the S-type lectin of microgram quantities of protein utilizing the principle of prot family.9) G. capensis lectin is quite different from congerin ein-dye binding. Anal. Biochem., 72, 248-254 (1976). because of the presence of cysteinyl residues in the amino 15) K. Muramoto and H. Kamiya: Recovery of tryptophan in peptides terminal region and its dependency of the hemagglutinat and proteins by high-temperature and short-term acid hydrolysis in ing activity on the calcium ion. These properties of G. the phenol. Anal. Biochem., 189, 223-230 (1990). 16) K. Muramoto, S. Sunahara, and H. Kamiya: Measurement of tryp capensis skin mucus lectin suggest that the lectin belongs tophan in peptides by acid hydrolysis in the presence of phenol and to the C-type lectin family. No notable homology, its application to the amino acid sequence of a sea anemone toxin. however, to the C-type lectins was observed in the search Agric. Biol. Chem., 51, 1607-1616 (1987). ing of sequence homology on a protein-sequence-data 17) K. Muramoto, R. Goto, and H. Kamiya: Analysis of reducing base. Therefore, the determination of the complete se sugars as their chromophoric hydrazones by HPLC. Anal. quence is necessary in order to group this new lectin into Biochem., 162, 435-442 (1987). the C-type lectin family. 18) Y. Oda, S. Ichida, T. Mimura, K. Tsujikawa, K. Maeda, and S. Aonuma. Mitogenic activity and in vitro fertilization inhibitiory ac In this study, we could not obtain any evidence or posi tivity of Genypterus blacodes lectin. J. Pharm. Dyn., 7, 849-855 tive result suggesting the participation of the skin mucus (1984).