Characterization of the Lectin from the Skin Mucus of the Kingklip Genypterus Capensis
Total Page：16
File Type：pdf, Size：1020Kb
Fisheries Science 62(1), 138-141 (1996) Characterization of the Lectin from the Skin Mucus of the Kingklip Genypterus 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 eels) 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 animal 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 eel 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.