Purification and characterization of a (1-->3)-beta-D-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes.
A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate. The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans. T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan. Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.
Tamura
H
H
Department of Biochemistry, Faculty of Science, Saitama University, Urawa, Japan.
Tanaka
S
S
Oda
T
T
Uemura
Y
Y
Aketagawa
J
J
Hashimoto
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005936:Glucans / Q000378:metabolism; D006737:Horseshoe Crabs; D037102:Lectins; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding
D002352:Carrier Proteins; D004731:Endotoxins; D005936:Glucans; D037102:Lectins; D008565:Membrane Proteins; C025357:endotoxin binding proteins; C037937:glucan-binding proteins; C014731:pachyman