Abstract
In this study, we report the isolation and characterization of three novel hemolymph proteins that are believed to be involved in the innate immune response of horseshoe crabs, Tachypleus tridentatus. They include two closely related proteins, one that binds to the protein A of Staphylococcus aureus (PAP) and another that binds to the lipopolysaccharide of Escherichia coli (LBP). PAP binds specifically to staphylococcal protein A (SpA) with a K(D) of 3.86 x 10(-5) M, whereas LBP binds to lipopolysaccharide (LPS) with a K(D) of 1.03 x 10(-6) M. Both PAP and LBP are glycoproteins with an apparent molecular mass of about 40 kDa. N-terminal sequences of PAP and LBP showed 61.9 and 72.2% identity, respectively, to tachylectin-3, a lectin isolated from the amebocyte of T. tridentatus, previously characterized by its affinity to the O-antigen of LPS and blood group A antigen (Muta, T., and Iwanaga, S. (1996) Curr. Opin. Immunol. 8, 41-47). The third protein, a galactose-binding protein (GBP), was found to bind tightly to Sepharose CL-4B and could only be eluted from the column matrix with chaotropic agents, such as 4 M urea or 2 M guanidine hydrochloride. Further analysis indicated that GBP binds to D(+)-galactose with a K(D) of 2.47 x 10(-7) M. N-terminal sequence analysis showed that GBP shared a 50% identity with lectin L-6, identified in the granules of amebocyte of T. tridentatus. (Gokudan, S., Muta, T., Tsuda, R., Koori, K., Kawahara, T., Seki, N., Mizunoe, Y., Wai, S. N. , Iwanaga, S., and Kawabata, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10086-10091). Lectin-L6 and tachylectin-3 are nonglycosylated intracellular proteins with about half the molecular mass of PAP, LBP, and GBP. GBP also binds to PAP and LBP with K(D) values of 1.25 x 10(-7) and 1.43 x 10(-8) M, respectively, and this binding is enhanced about 10-fold upon the addition of SpA and LPS to form the GBP.PAP.SpA and GBP.LBP.LPS complexes, respectively.
Highlights
Invertebrates have developed several germ line-encoded receptor-dependent pathways, such as proteolytic and prophenol oxidase cascade, pathogen-specific lectins, and antibiotic peptides, which work in concert for the recognition, immobiliza
C-reactive proteins are lectins that bind to phosphocholine of the pneumonococcus C-polysaccharide [7] and to the chromatins of damaged cells [8]. ␣2-macroglobulin exhibits protease inhibitory activity with a broad specificity that can block the activities of proteases secreted from invading microorganisms [9, 10]
Purification of Three Hemolymph Lectins—By passing hymolymph of horseshoe crabs through a tandemly linked affinity columns, we have purified and characterized three new lectins: a galactose-binding protein (GBP) that binds to the galactose matrix of Sepharose CL-4B, a protein A of Staphylococcus aureus (PAP) that binds to the staphylococcal protein A (SpA) of Staphylococcus aureus, and a LBP that binds to the LPS of E. coli
Summary
Invertebrates have developed several germ line-encoded receptor-dependent pathways, such as proteolytic and prophenol oxidase cascade, pathogen-specific lectins, and antibiotic peptides, which work in concert for the recognition, immobiliza-. The hemolymph of horseshoe crab contains three major proteins: hemocyanin, C-reactive protein, and ␣2-macroglobulin. Several lectins have been identified in the amebocytes of horseshoe crab, with a broad range of specificity [2, 12, 13]. These lectins have been proposed to function in concert to defend horseshoe crabs from invading pathogens. Because these lectins are present in the granules of the hemocytes, they are unlikely to be involved in the immediate-early response of host-pathogen interaction. A model of interaction for these proteins has been proposed
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