Isolation and characterization of proteins that bind to galactose, lipopolysaccharide of Escherichia coli, and protein A of Staphylococcus aureus from the hemolymph of Tachypleus tridentatus

In this study, we report the isolation and characterization of three novel hemolymph proteins that are believed to be involved in the innate immune re sponse 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 affin ity to the O-antigen of LPS and blood group A antigen (Muta, T., and Iwanag a, 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 co uld only be eluted from the column matrix with chaotropic agents, such as 4 iu urea or 2 nr guanidine hydrochloride. Further analysis indicated that G BP binds to D(+)-galactose with a K-D of 2.47 x 10(-7) M. N-terminal sequen ce analysis showed that GBP shared a 50% identity with lectin L-6, identifi ed 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,, Iwan aga, S., and Kawabata, S. (1999) Proc. Natl. Acad Sci. U.S.A 96, 10086-1009 1). 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, respectiv ely, and this binding is enhanced about 10-fold upon the addition of SpA an d LPS to form the GBP.PAP SpA and GBP.LBP.LPS complexes, respectively.