Purification, Partial Biological Function And Gene Cloning Of β-1, 3-Recognition Protein From Tenebrio Molitor

Although several pattern recognition proteins have been identified in vertebrates and insects, the molecular mechanism and structural basis of microbial recognition are largely unknown. A key question of invertebrates prophenoloxidase (pro-PO) activation system is how pattern recognition, like peptidoglycan orβ-1,3-glucan recognition molecules can induce activation of the pro-PO system in response to pathogenic microbial infection.To determine the biological functions ofβ-1,3-glucan recognition protein in the insect pro-PO activation system, A novelβ-1,3-glucan recognition protein (Tm-βGRP) was purified to homogeneity from the hemolymph of mealworm, Tenebrio molitor, by usingβ-1,3-glucan affinity column. The purified protein specifically bound toβ-1,3-glucan but not peptidoglycan, and this protein was localized in plasma. The experimental results of functional research shown, the amounts of endogenous Tm-βGRP during pro-PO activation was specifically degraded and the polycloning antibody inhibited the activation of pro-PO system in a dose dependent manner. After adding the exogenous Tm-pGRP to crude hemolymph, the activation of pro-PO system was enhanced. The results of in vitro reconstitution experiment with fractions of affinity chromatography shown, there has another essential component of pro-PO activation system can bind withβ-1,3-glucan specially except Tm-βGRP.Molecular cloning results provided that Tm-PGRP contained an open reading frame with 1443 nts and encoded to one protein with 481 amino acid residues, which assumed molecular weight was 54034.2 Da and the predicated pI was 7.545. According to the results of sequence alignment of Tm-βGRP, this protein shown high homology withβ-1,3-glucan recognition proteins (βGRPs) and some Gram negative bacterial binding proteins (GNBPs) in other insectsOn the summary, this research found a new protein-Tm-βGRP, which bound withβ-1,3-glucan specially and actived the pro-PO activation system in Tenebrio molitor larvae. We demonstrated two novel findings regarding a glucan-binding protein from invertebrate. One was that the amount of Tm-βGRP was decreased when PO activity was triggered by β-1,3-glucan and the other was that there had another essential component of pro-PO activation system, which can bind withβ-1,3-glucan specially except Tm-βGRP.