Abstract
Glycan-binding proteins (lectins) are widely expressed in many invertebrates, although the biosynthesis and functions of the lectins are not well understood. Here we report that Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) upon infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a approximately 74-kDa precursor and secreted into hemolymph where it is converted to 30- and 34-kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by one or more factors in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite, and this binding is inhibitable by either EDTA or GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL is concentrated within cyst-like structures. To define the glycan binding specificity of MCL we examined its binding to an array of biotinylated glycans. MCL recognizes terminal non-reducing beta-linked GalNAc as expressed within the LacdiNAc motif GalNAcbeta1-4GlcNAcbeta1-R and glycans with terminal, non-reducing beta-linked Gal residues. Our results show that the synthesis of MCL is specifically up-regulated upon parasite infection of the clams and may serve as an opsonin through recognition of terminal GalNAc/Gal residues on the parasites.
Highlights
These lectins may be classified into two general groups
We report that the infection of clams by the protozoan parasite Perkinsus olseni induces the synthesis of Manila clam lectin (MCL) by clam hemocytes
Induction of MCL upon Perkinsus Infection—MCL was purified from Manila clams as described previously [26]
Summary
Purification of Perkinsus Trophozoites from Manila Clam— Trophozoites of P. olseni were extracted from the hemolymph withdrawn from the blood sinus of the adductor muscle of Manila clams, which were collected at Wando Island off the southern coast of Korea, where a high incidence of the parasite infection has been reported [27]. An equal volume of 6% neutral formalin was added to the fix hemocytes, which were analyzed by flow cytometry Another control group was prepared where beads that had been incubated with 1 ml of filtered seawater were added to the hemocyte suspension and immediately mixed with 3 l of cytochalasin B at 0.1% final concentration to block further phagocytosis. The membrane was washed three times for 10 min each in TTBS and incubated with anti-MCL antibody (1:500 dilution in TTBS plus 1% bovine serum albumin) for 1 h at room temperature. The cells were washed three times in PBS-T and incubated with a 1:200 dilution in PBS-T of FITC-conjugated goat antibody against rabbit immunoglobulins (Sigma-Aldrich) for 1 h. Manual assignments of fragment ions in each MS/MS spectra were performed to confirm the protein data base search results
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