A novel C-type immulectin-3 from Manduca sexta is translocated from hemolymph into the cytoplasm of hemocytes.
Lectins interact with carbohydrates. They can function as pattern recognition receptors and play an important role in the innate immune system of animals. Previously, we have isolated two calcium-dependent (C-type) lectins, named immulectin-1 and -2, from the tobacco hornworm Manduca sexta. Both immulectin-1 and -2 stimulate prophenoloxidase activation in plasma. Here, we describe isolation and cDNA cloning of a novel member of immulectins, immulectin-3 (IML-3). IML-3, like immulectin-1 and -2, contains tandem carbohydrate-recognition domains (CRDs). The cDNA clone encoding IML-3 is 3802 bp long, with an open reading frame of 930 bp. This cDNA clone has an extremely long noncoding region at the 3' end that contains eight polyadenylation signal sequences. Northern analysis showed that a 5.0 kb IML-3 transcript was present in the fat body of control larvae (injected with saline) but not in the fat body of larvae injected with bacteria. However, a much more abundant 3.1 kb transcript was induced in the fat body of bacteria-injected larvae. IML-3 mRNA was not detected in hemocytes of control or bacteria-injected larvae. Recombinant IML-3 was expressed in bacteria and purified. It specifically bound to immobilized lipopolysaccharide (LPS) and lipoteichoic acid from bacteria, and to laminarin, a beta-1, 3-glucan. Binding of IML-3 to immobilized LPS was competed by excess free LPS. More importantly, IML-3 contains an anti-death-like motif in the carboxyl-terminal CRD. Endogenous IML-3 was detected in the cytoplasm of hemocytes, and FITC-labeled recombinant IML-3 was translocated from hemolymph into hemocytes. Coating of IML-3 onto agarose beads enhanced encapsulation of the beads.
Yu
Xiao-Qiang
XQ
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA. yux@umkc.edu
Tracy
Miles E
ME
Ling
Erjun
E
Scholz
Frank R
FR
Trenczek
Tina
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D006434:Hemocytes / Q000378:metabolism*; D006458:Hemolymph / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D018402:Manduca / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D019476:Insect Proteins; D037181:Lectins, C-Type; D011994:Recombinant Proteins