Biological Effects of a Carbohydrate-Binding Protein from an Annelid, Perinereis nuntia Against Human and Phytopathogenic Microorganisms

Lectins have a good scope in current clinical microbiology research. In the present study evaluated the antimicrobial activities of a D-galactose binding lectin (PnL) was purified from the annelid, Perinereis nuntia (polychaeta) by affinity chromatography. The molecular mass of the lectin was determined to be 32 kDa as a single polypeptide by SDS-PAGE under both reducing and non-reducing conditions. The hemagglutinating activity of the PnL showed against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-Gal, GalNAc, Galβ1-4Glc and Galα1-6Glc. PnL was evaluated for in vitro antibacterial screening studies against 11 gram-positive and gram-negative microorganisms. From the screening results, it was revealed that PnL exhibited significant antibacterial activity against gram-positive bacteria. Bacillus megaterium showed the highest growth inhibition by the lectin (250 μg/disc). However, PnL did not inhibit the growth of gram-negative bacteria such as Vibrio cholerae and Pseudomonas sp. PnL was also examined for in vitro antifungal activity against six fungal phytopathogens. PnL (100 μg/mL) inhibited the mycelial growth of Alternaria alternata (24.4%). These results indicate that future findings of lectin applications obtained from annelids may be of importance to life sciences.

Authors:

• Sarkar M. A. Kawsar
• Sarkar M. A. Mamun
• Md S. Rahman
• Hidetaro Yasumitsu
• Yasuhiro Ozeki

Keywords:

• Perinereis nuntia
• Lectin
• Inhibition zone
• Mycelial growth.

References:

[1] Kilpatrick, D. C. (2002). Animal lectins: an historical introduction and
over-view. Biochim Biophys Acta, 1572, 187-197.
[2] Vazquez, L., Alpuche, J., Maldonado, G., Agundis, C., Morales, A. P.,
and Zenteno, E. (2009). Immunity mechanisms in crustaceans. Innate
Immun, 15, 179-188.
[3] Gabius, H. J., Unverzagt, C., and Kayser, K. (1998). Beyond plant lectin
histochemistry: preparation and application of markers to visualize the
cellular capacity for protein-carbohydrate recognition. Biotech Histochem
73, 263-277.
[4] Stabili, I., Pagliara, P., and Roch, P. (1996). Antibacterial activity in the
coelomocytes of the sea urchin Paracentrotus lividus. Comp Biochem
Physiol, 113B, 639-644.
[5] Ozeki, Y., Tazawa, E., and Matsui, T. (1997). D-Galactoside-specific
lectins from the body wall of an echiuroid (Urechis unicinctus) and two
annelids (Neanthes japonica and Marphysa sanguinea). Comp Biochem
Physiol, 118B, 1-6.
[6] Garte, S. J., and Rissell, C. S. (1976). Isolation and characterization of a
hemagglutinin from Amphitrite ornate, a polychaetous annelid. Biochem
Biophys Acta, 439, 368-379.
[7] Molchanova, V., Chikalovets, I., Chernikov, O., Belogortseva, N., Li, W.,
Wang, J. H., Yang, D. Y. O., Zheng, Y. T., and Lukyanov, P. (2007). A
new lectin from the sea worm Serpula vermiculasis: isolation,
characterization and anti-HIV-1 activity. Comp Biochem Physiol, 145C,
184-193.
[8] Wang, J. H., Kong, J., Li, W., Molchanova, V., Chikalovets, I.,
Belogortseva, N., Lukyanov, P., and Zheng, Y. T. (2006). A
β-galactose-specific lectin isolated from the marine worm Chaetopterus
variopedatus possesses anti-HIV-1 activity. Comp Biochem Physiol,
142C, 111-117.
[9] Mikheyskaya, L. V., Evtushenko, E. V., Ovodova, R. G., Belogortseva, N.
I., and Ovodov, Y. S. (1995). Isolation and characterization of a new
β-galactoside-specific lectin from the sea worm Chaetopterus
variopedatus. Carbohydr Res, 275, 193-200.
[10] Hirabayashi, J., Dutta, S. K., and Kasai, K. (1998). Novel
galactose-binding proteins in Annelida. Characterization of 29-kDa
tandem repeat-type lectins from the earthworm Lumbricus terrestris. J
Biol Chem, 273, 14450-14460.
[11] Cole, R. N., and Zipser, B. (1994). Carbohydrate-binding proteins in the
leech: I. Isolation and characterization of lactose-binding proteins. J
Neurochem, 63, 66-74.
[12] Wang, H. X., Liu, W. K., Ng, T. B., Ooi, V. E., and Chang, S. T. (1996).
The immunomodulatory and antitumor activities of lectins from the
mushroom Tricholoma mongolicum. Immunopharmacol, 31, 205-211.
[13] Yu, L. G., Milton, J. D., and Fernig, D. G. (2001). Opposite effects on
human colon cancer cell proliferation of two dietary
Thomsen-Friedenreich antigen-binding lectins. J Cell Physiol, 186,
282-287.
[14] Singh, B. J., Sing, J., Kamboj, S. S., Nijar, K. K., Agrewale, J. N., Kumar,
V., Kumar, A., and Saxena, A. K. (2005). Mitogenic and anti-proliferative
activity of a lectin from the tubers of Voodoo lily (Sauromatum venosum).
Biochim Biophys Acta, 1723, 163-174.
[15] Ye, X. Y., Ng, T. B., Tsang, P. W., and Wang, J. (2001). Isolation and
homodimeric lectin with antifungal and antiviral activities from red
kidney bean (Phaseolus vulgaris) seeds. J Protein Chem, 20, 367-375.
[16] Ooi, L. S., Ng, T. B., Geng, Y., and Ooi, V. E. (2000). Lectins from bulbs
of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae).
Biochem Cell Biol, 78, 463-468.
[17] Machuka, J. S., and Oladapo, G. (2000). The African yam bean seed lectin
affects the development of the cowpea weevil but does not affect the
development of larvae of the legume pod borer. Phytochem, 53, 667-674.
[18] Cotuk, A., and Dales, R. P. (1984). Lysozyme activity in the coelomic
fluid and coelomocytes of the earthworm Eisenia foetida Sav. In relation
to bacterial infection. Comp Biochem Physiol, 78A, 469-474.
[19] Ito, Y., Yoshikawa, A., Hotani, T., Fukuda, S., Sugimura, K., and Imoto,
T. (1999). Amino acid sequences of lysozymes newly purified from
invertebrates imply wide distribution of a novel class in the lysozyme
family. Eur J Biochem, 259, 456-461.
[20] Tunkijjanukij, S., and Olafsen, J. A. (1998). Sialic acid-binding lectin
with antibacterial activity from the horse mussel: further characterization
and immunolocalization. Dev Comp Immunol, 22, 139-150.
[21] Olafsen, J.A. (1995). Role of lectins (C-reactive protein) in defense of
marine bivalves against bacteria.
Adv Exp Med Biol, 371: 343-348.
[22] Gowda, N. M., Goswami, U., and Khan, M. I. (2008). T-antigen binding
lectin with antibacterail activity from marine invertebrate, sea cucumber
(Holothuria scabra): possible involvment in differential recognition of
bacteria. J Invertebr Pathol, 99, 141-145.
[23] Liu, Y. Q., Sun, Z. J., Wang, C., Li, S. J., and Liu, Y. Z. (2004).
Purification of a novel antibacterial short peptide in earthworm Eisenia
foetida. Acta Biochim Biophys Sinica. 36, 297-302.
[24] Dhainaut, A., Raveillon, B., M-Beri, M., Hennere, P. E., and Demuynck,
S. (1989). Purification of an antibacterial protein in the coelomic fluid of
Nereis diversicolor (annelida, polychaeta). Similitude with a
cadmium-binding protein. Comp Biochem Physiol. 1989; 94C: 555-560.
[25] Tasiemski, A., Schikorski, D., Croq, F. L. M., Camp, C. P. V., Wichlacz,
C. B., and Sautiere, P. E. (2007). Hedistin: A novel antimicrobial peptide
containing bromotryptophan constitutively expressed in the NK cells-like
of the marine annelid, Nereis diversicolor. Dev Comp Immunol, 31,
749-762.
[26] Sato, Y., Okuyama, S., and Hori, K. (2007). Primary structure and
carbohydrate binding specificity of a potent anti-HIV lectin isolated from
the filamentous cyanobacterium Oscillatoria agardhii. J Biol Chem, 282,
11021-11029.
[27] Kawsar,, S. M. A., Takeuchi, T., Kasai, K-I., Fujii, Y., Matsumoto, R.,
Yasumitsu, H., and Ozeki, Y. (2009). Glycan-binding profile of a
D-galactose binding lectin purified from the annelid, Perinereis nuntia
ver vallata. Comp Biochem Physiol, 152B, 382-389.
[28] Matsui, T. (1984). D-galactoside specific lectins from coelomocytes of
the echiuran, Urechis unicinctus. Biol Bull, 178-188.
[29] Laemmli, U. K. (1970). Cleavage of structural proteins during assembly
of the head of bacteriophage T4. Nature, 227, 680-685.
[30] Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H.,
Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and
Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid.
Anal Biochem, 150, 76-85.
[31] Wiechelman, K. J., Braun, R. D., and Fitzpatrick, J. D. (1988).
Investigation of the bicinchoninic acid protein assay: identification of the
groups responsible for color formation. Anal Biochem. 175, 231-237.
[32] Bauer, A. W., Kirby, M. M., Sherris, J. C., and Turck, M. (1966).
Antibiotic susceptibility testing by a standardized single disc method. Am
J Clin Path, 45, 493-496.
[33] Grover, R. K., and Moore, J. D. (1962). Toximetric studies of fungicides
against the brown rot organisms, Sclerotinia fructicola and S. laxa
.Phytopathology, 52, 876-880.
[34] Miah, M. A. T., Ahmed, H. U., Sharma, N. R., Ali, A., and Miah, S. A.
(1990). Antifungal activity of some plant extracts. Bang J Bot, 19, 5-10.
[35] Oliveira, M. D. L., Andrade, C. A. S., Magalhaes, N. S. S., Coelho, L. C. B.
B., Teixeira, J. A., Cunha, M. G. C., and Correia, M. T. S. (2008).
Purification of a lectin from Eugenia uniflora L. seeds and its potential
antibacterial activity. Appl Microbiol, 46, 371-376.
[36] Naganuma, T., Ogawa, T., Hirabayashi, J., Kasai, K., Kamiya, H., and
Muramoto, K. (2006). Isolation, characterization and molecular evolution
of a novel pearl shell lectin from a marine bivalve, Pteria penguin. Mol
Div, 10, 607-618.
[37] Tateno, H., Ogawa, T., Muramoto, K., Kamiya, H., and Saneyoshi, M.
(2002). Rhamnose-binding lectins from steelhead trout (Oncorhynchus
mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic
acid. Biosci Biotechnol Biochem, 66, 604-612.
[38] Calderon, A. M., Buck, G., and Doyle, R. J. (1997).
Lectin-microorganism complexes. In Lectins, Biology, Biochemistry,
Clinical Biochemistry Vol 12 ed. van Driessche, E., Beeckmans, S.,
B├©g-Hansen, T. C., and Lemchesvej, Hellerup, Denmark: TEXTOP.
http://plab.ku.dk/tcbh/Lectins12/Calderon/paper.htm.
[39] Munoz-Crego, A., Alvarez, O., Alonso, B., Rogers, D. J., and Lvovo, J.
(1999). Lectin as diagnostic probes in clinical bacteriology-an overview.
In Lectins, Biology, Biochemistry, Clinical Biochemistry Vol 13 ed. van
Driessche, E., Beeckmans, S., B├©g-Hansen, T. C., and Lemchesvej,
Hellerup, Denmark: TEXTOP.
http://plab.ku.dk/tcbh/Lectins12/Calderon/paper.htm.
[40] Doyle, R. J. (1994). Introduction to lectins and their interactions with
microorganisms. In Lectin-microorganism Interactions ed. Doyle RJ,
Slifkin MV, New York, Marcel Dekker, Inc. pp. 1-65.
[41] Nagi, P. H. K., and Ng, T. B. (2007). A lectin with antifungal and
mitogenic activities from red cluster pepper (Capsicum frutescens) seeds.
Appl Microbiol Biotechnol, 74, 366-371.
[42] Sitohy, M., Doheim, M., and Badr, H. (2007). Isolation and
characterization of a lectin with antifungal activity from Egyptian Pisum
sativum seeds. Food Chem, 104, 971-979.
[43] Broekaert, W. F., Van, P. J., Leyn, F., Joos, H., and Peumans, W. (1998).
A chitin-binding lectin from stinging nettle rhizomes with antifungal
properties. Science, 245, 1100-1102.
[44] Dhainaut, A., and Scaps, P. (2001). Immune defense and biological
responses induced by toxics in Annelida. Can J Zool, 79, 233-253.
[45] Paul, V. J., and Puglisi, M. P. (2004). Chemical mediation of interactions
among marine organisms. Nat Prod Rep, 21, 189-209.
[46] Kelly, S. R., Garo, E., Jensen, P. R., Fenical, W., and Pawlik, J. R. (2005).
Effects of Caribbean sponge secondary metabolites on bacterial surface
colonization. Aquat Microb Ecol, 40, 191-203.