Characterization and functional study of hemolin and a novel C-type lectin from the tobacco hornworm, Manduca sexta

Insect immunity is similar to vertebrate innate immunity, but little is known about recognition of nonself by insects. In this research, hemolin and a C-type lectin from the tobacco hornworm Manduca sexta, were identified as potential recognition molecules.; Hemolin, a member of the immunoglobulin superfamily, is a bacteria-inducible protein in hemolymph of M. sexta larvae. I have discovered that hemolin is also present without bacterial challenge in plasma of wandering stage larvae, pupae, and adults, and in the midgut lumen of prepupae and pupae. Hemolin from these developmental stages was purified and characterized. Hemolin from bacteria-induced larvae and adults contained non-covalently bound carbohydrates. Hemolins from all developmental stages have a molecular mass of 44 kDa (determined by mass spectrometry), the size predicted from hemolin cDNA. They have identical amino terminal sequence, except for adult hemolin, which was blocked. Hemolin bound to bacteria and yeast, and agglutinated them in a concentration-dependent manner. LPS inhibited the agglutination of bacteria by hemolin. These results suggest that hemolin functions as a recognition molecule.; A novel C-type lectin (LPS-binding lectin, LBL) cDNA from M. sexta was isolated. LBL is a 33.4 kDa protein with a carboxyl-terminal carbohydrate recognition domain similar to LPS-binding protein from the American cockroach and to rat mannose-binding protein. The amino-terminal half of LBL is not similar to any other known sequence. LBL synthesis was induced in larvae by injection of bacteria or rabbit red blood cells. A 1.4 kb LBL mRNA was induced in fat body after injection of bacteria. Two LBL isoforms were identified in plasma. Southern blot analysis showed that there are probably two related lectin genes. Recombinant LBL was expressed in E. coli and in insect cells. LBL agglutinated bacteria and yeast. LPS inhibited agglutination of bacteria by LBL. LBL activated hemolymph phenoloxidase. A combination of LPS and LBL activated phenoloxidase more rapidly and to a higher level than LBL alone. These results suggest that LBL is involved in a serine protease cascade triggered by LPS, leading to activation of phenoloxidase, which resembles activation of the complement pathway by mammalian mannose-binding protein.