Griffonia simplicifolia

About the Lectin

The seeds of Griffonia simplicifolia contain four distinct lectins. Two of these lectins, referred to as GS-I and GS-II, have been well characterized with regard to binding specificity and structure. GS-I exists as a mixture of five isolectins. Each isolectin is a tetramer consisting of different amounts of two subunits, termed A and B 1 . The five isolectins have the following general forms: A 4 , A 1 B 3 , A 2 B 2 , A 3 B, B 4 . The subscripted numbers indicate the quantity of each subunit associated with the tetrameric form. Comparative binding studies have been performed using the A 4 and B 4 isolectins. The B 4 isolectin has a specificity for type B erythrocytes, while the A 4 isolectin has a specificity for type A cells. Each of the two isolectins has an equal affinity for a-galactosyl end groups, however the A 4 isolectin has a greater affinity for α-GalNAc than the B 4 isolectin. A specific anti-B lectin can be produced by the addition of GalNAc to the GS-I preparation 2 . The abundance of the individual isolectins will vary from seed to seed, and potentially from batch to batch, depending on the purification method used. Purified GS-I-B 4 has been shown to be cytotoxic to Swiss 3T3 mouse cells and to Ehrlich ascites tumor cells 3 . Stimulated mouse macrophages express a carbohydrate determinant that will react specifically with GS-I-B 4 . This determinant is not present on unstimulated cell populations 4 . GS-I-B 4 also reacts with laminin 5 isolated from the mouse EHS sarcoma, and with thyroglobulin 6 .

Immobilized GS-I-B 4 was employed to resolve oligosaccharides containing α-D-galactosyl 7 , and to isolate Gal α1-3Gal-containing end groups from calf thyroid cells. 8 The tissues from a normal 9 and an &#945-1,3-galactosyltransferase knockout mouse 10 were scanned using biotinylated 10 and fluorescein-labeled B 4 9 . Nucleotide sugars and oligosaccharides bearing &#945-D-GalNAc end groups were resolved by affinity chromatography on immobilized GS I-A4.11 The A4 isolectin was also shown to bind to the Tn antigen (&#945-GalNAc-O-Ser/Thr) in humans with colon cancer, and to colon tissue from familial adenomatous polyopsis; it bound and was cytotoxic to two colon cancer cell lines, much more so often than other GalNAc-specific lectins. 12

GS-II is specific for terminal, non-reducing α- or β-linked N-acetylDglucosamine. This lectin is not blood group specific, unlike GS-I. GS-II is also a tetramer but it is composed of identical subunits. Each subunit has a single carbohydrate binding site for GlcNAc. GS-II is the only lectin isolated that is specific for only a terminal GlcNAc residue. The subterminal saccharide does play an important role in lectin binding. GlcNAc linked &#946(1,3) or &#945(1,6) to galactose is a poor inhibitor of the lectin while GlcNAc linked α(1,3) to galactose or glucose is a potent inhibitor 13 . The GS-II lectin has been used as a probe for the isolectin GlcNAcβ1-3Galβ1-4GlcNAc sequence in tissue sections following treatment with endo-β-galactosidase. 14 Glycogen in tissue sections was demonstrated in man and rodents using a GS-II-horseradish peroxidase system. 15

GS-II has insecticidal activity against cowpea weevil. 16 A cDNA clone encoding GS-II was isolated, sequenced and expressed in a bacterial expression system; the recombinant protein bound GlcNAc and had insecticidal activity against the weevil. The crude form of the lectin contains both the GS-I isolectins and the GS-II lectin.

REFERENCES

  1. Murphy, L. A. and Goldstein, I. J. (1977). J. Biol. Chem.252 : 4739-4742.
  2. Judd, W. J., et al. (1978). Transfusion (Philadelphia).18 : 274-280.
  3. Eckhardt, A. E., et al. (1982). Cancer Res. 42 : 2977-2979.
  4. Maddox, D. E., et al. (1982). PNAS. 79 : 166-170.
  5. Knibbs, R.N., et al. (1989) Biochemistry 28 : 6379-6392.
  6. Spiro, R.G. and Bhoyroo, V.D. (1984) J. Biol. Chem. 259 : 9858-9866.
  7. Wang, W.C.,et al. (1988) Anal. Biochem. 175 : 390-396.
  8. Edge, A.S.B. and Spiro, R. G. (1997) Arch, Biochem. Biophys. 343 : 73-80.
  9. Peters, B.P. and Goldstein, I.J. (1979) Exp. Cell Res. 120 : 321-334.
  10. Tearle, R.G., et al. (1996) Transplantation 61 : 13-19.
  11. Blake, D.A. and Goldstein, I.J. (1980) Anal. Biochem. 102 : 103-109.
  12. Chen, Y.-F., et al. (1994) Int. J. Cancer 57 :561-567.
  13. Shanker Iyer, P. N., et al. (1976). Arch. Biochem. Biophys.177 : 330-333.
  14. Kawahara, I.N., et al. (1994) Histochem. J. 26 : 327-336.
  15. Hennigar, R.A., et al. (1986) Histochem. J. 18 : 589-596.
  16. Zhu, K., et al. (1996) Plant Physiol. 110 : 195-202.

Product Characteristics – GS I

Buffer 0.01M Phosphate – 0.15M NaCl containing 0.5mM CaCl2, pH 7.2-7.4
Blood Group B erythrocytes. Slight reactivity with A1 cells, evidently due to the A subunit.
Activity 20-30 μg/ml is required to agglutinate fresh type B blood cells.
The isolated GS-I-B4 lectin will react stronger than the isolectin mixture.
Lectin activity against all blood types increases after neuraminidase treatment of the cells.
Inhibitory Carbohydrate α-D-Galactoside and α-linked galactose oligosaccharides.
N-acetyl‑D‑galactosamine is an inhibitor of the A subunit, only.
Molecular Weight Aggregate MW=114,000. Two bands between MW=28-32,000 appear on SDS-PAGE.

Product Characteristics – GS II

Buffer 0.01M Phosphate – 0.15M NaCl containing 0.5mM CaCl2, pH 7.2-7.4
Blood Group Non-reactive with normal erythrocytes.
GS-II does agglutinate acquired-B, T-activated, and Tk polyagglutinable cells.
Activity 5-10 μg/ml will agglutinate Tk polyagglutinable cells.
Inhibitory Carbohydrate N-acetyl‑D‑glucosamine.
Molecular Weight Aggregate MW=113,000. Single band of MW=30,000 by SDS-PAGE.