Lectin from the Body Walls of Black Sea Cucumber (Holothuria atra Jaeger)
Elmer-Rico E. Mojica and Florinia E. Merca
Institute of Chemistry, College of Arts and Sciences, University of the Philippines
Los Baños, College, Laguna
A survey of lectin activity was made on the crude extracts of 15 species of locally available marine invertebrates. Thirteen species were found to show lectin activity. The body wall of Holothuria atra Jaeger, which had the highest lectin activity, was chosen for further examination. Lectin was extracted from the body walls of black sea cucumber (Holothuria atra Jaeger) using Tris buffer saline (TBS) pH 7.5 containing 0.15 M NaCl and purified by ammonium sulfate fractionation and gel chromatography using Sephadex G-200. The isolated lectin was non-blood type specific as it agglutinated erythrocytes of all human blood types (A, B, AB, O) and animal erythrocytes (calf, carabao, chicken and goat). Addition of trypsin and calcium ions increased the agglutinating activity of both lectins. The isolated lectin was found to be glycoprotein containing 0.50% total sugar. Based on gel chromatography, the estimated molecular weight of lectin from the body wall is 439 kD. SDS-PAGE gave two protein bands for the isolated lectin with molecular weights estimated to be 113 kD and 77 kD .
Lectins are carbohydrate-binding proteins or glycoproteins of non-immune origin, which are able to agglutinate cells and precipitate polysachaccharides and glycoconjugates (Goldstein et al. 1980). They are of great interest because of various applications. Their carbohydrate-binding specificity enables their use in cell separation techniques, in isolation and purification procedures in biochemistry, cell biology, pharmacology, immunology and other related areas. It has beenused in the isolation, purification and structural studies of carbohydrate-. . . . . .
[AOAC] Association of Official Analytical Chemists. 1993. Official methods of analysis. 16th ed. Washington, DC: Association of Official Analytical Chemists, Inc.
BRADFORD MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities for protein using the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.
BROWN R, ALMODOVAR LR, BHATIA HM, BOYD WC. 1968. Blood group specific agglutinins in invertebrates. Journal of Immunology 100: 214-216.
BULGAKOV AA, NAZARENKO EL, PETROVA IY, ELISEIKINA MG, VAKHRUSHEVA NM, ZUBKOV VA. 2000. Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica. Biochemistry (Moscow) 65(8): 933-939.
COOPER TG. 1971. The Tools of Biochemistry. New York: John Wiley and Sons. Drickamer K. 1988. Two distinct classes of carbohydraterecognition domains in animal lectins. Journal of Biological Chemistry 263: 9557-60.
DUBOIS M, GILLES KA, HAMILTON JK, REBERS PA, SMITH F. 1956. Phenol-sulfuric acid method for determining total sugars. Analytical Chemistry 28: 350-356.
GANA AE, MERCA FE. 2002. Isolation and purification of a cytotoxic lectin from brown sea cucumber by affinity chromatography. Philippine Agricultural Scientist 85(3): 236-247.
GIGA Y, IKAI A, TAKAHASHI K. 1987. The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins. Journal of Biological Chemistry 262(13): 6197-6203.
GOLDSTEIN I, HAYES RC, MONSUGNY M, OSAWA T, SHARON N. 1980. What should be called a lectin? Nature 285: 66.
GORDON JA, SHARAON N, LIS H. 1972. Binding of soybean agglutinin by normal and trypsin-treated red blood cells. Biochimica Biophysica Acta 264: 387-391.
HAGEL L. 1989. Gel filtration. In: Protein Purification Principles, High Resolution Methods and Applications. Janson JC and Ryden L (eds). VCH Publishers.
HATAKEYAMA T, HIMESHIMA T, KOMATSU A, YAMASAKI N. 1993. Purification and characterization of two lectins from the sea cucumber Stichopus japonicus. Bioscience, Biotechnology and Biochemistry 57(10): 1736-9.
HATAKEYAMA T, KOHZAKI H, NAGATOMO H, YAMASAKI N. 1994. Purification and characterization of four Ca2+- dependent lectins from the marine invertebrates Cucumaria echinata. Journal of Biochemistry 116: 209-214.
HATAKEYAMA T, OHUCHI K, KUROKI M, YAMASAKI N. 1995. Amino acid sequence of a C-type lectin CEL-IV from the marine invertebrate, Cucumaria echinata. Bioscience, Biotechnology and Biochemistry 59: 1314-17.
KIMURA T, IMAI Y, IRIMURA T. 1995. Calciumdependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calciumdependent epitope. Journal of Biological Chemistry 270(27): 16056-62.
LACSAMANA MS, MERCA FE. 1994. Isolation and characterization of a lectin from the visceral region of the golden snail, Pomacea sp. Philipp. J Sci. 123(1): 77-97.
LAEMMLI UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
LIS H, SHARON H. 1986. Applications of lectins and biological properties of lectins. In: the Lectins: properties, functions and applications in biology and medicine. Liener IE, Sharon N and Goldstein I (eds.) USA: Academic Press, Inc. p. 265-370.
MATSUI T, OZEKI Y, SUSUKI M, HINO A, TITANI K 1994. Purification and characterization of two Ca(2+)-dependent lectins from coelomic plasma of sea cucumber, Stichopus japonicus. Journal of Biochemistry 116: 1127-33.
MERCA FE, REYES LB. 1989. Isolation and partial characterization of a lectin from Ampullaria luzonica Reeve. Philipp. J Sci. 118: 307-322.
MURRAY RD, GRANNER D, MAYES P, RODWELL V. 1990. Harper’s Biochemistry. Prentice Hall International Inc.
MUTA T, MIYATA T, MISUMI Y, TOKUNAGA F, NAKAMURA T, TOH Y, IKEHARA Y, IWANAGA S. 1991. Limulus factor C. Journal of Biological Chemistry 266: 6554-61.
NAISMITH JH, FIELD RA. 1996. Structural basis of trimannoside recognition by Con A. Journal of Biological Chemistry 271(2): 972-976.
NICOLSON G. 1971. Difference in topology of normal and tumour cell membranes as shown by different surface distributions of ferritin conjugated Concanavalin A. Nature New Biology 233: 244.
SEGREST JP, JACKSON RL. 1972. Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. p. 54-62. In: Methods in Enzymology. V 28. Part B. V. Ginsburg (ed.). New York: Academic Press, Inc. 1057p.
SHARON N, LIS H. 1974. Glycoproteins. Sci Am 230: 78-86.
SHARON N, LIS H. 1987. A century of lectin research. TIBS 12: 488-491.
SINGH J, KAMBOJ KK, KAMBOJ SS, SANDHU RS, SHANGARY S. 1993. Affinity purification and characterization of lectins from two Amaranthus species. Plant Science 94: 47-53.
TAKAGAI T, NAKAMURA A, DEGUCHI R, KYOSUKA K. 1994. Isolation, characterization, and primary structure of three major proteins obtained from Mytilus edulis sperm. Journal of Biochemistry 116: 598-605.
TSIVION Y, SHARON N. 1981. Lipid mediated hemagglutination and its relevance to lectinmediated agglutination. Biochimica Biophysica Acta 642: 336-344.
WEIS WI, DRICKKAMER K, HENDRICKSON WA. 1992. Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 360: 127-134.
WITTENBACK VA. 1983. Purification and characterization of a soybean leaf storage glycoprotein. Plant Physiology 73: 125-129.