Daisuke Iwaki

Surfactant Proteins A and D Bind CD14 by Different Mechanisms

Surfactant proteins A (SP-A) and D (SP-D) are lung collectins that are constituents of the innate immune system of the lung. Recent evidence (Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D. R., and Kuroki, Y. (1999) J. Immunol. 163, 387–395) demonstrates that SP-A modulates lipopolysaccharide (LPS)-induced cellular responses by direct interaction with CD14. In this report we examined the structural elements of the lung collectins involved in CD14 recognition and the consequences for CD14/LPS interaction. Rat SP-A and SP-D bound CD14 in a concentration-dependent manner. Mannose and EDTA inhibited SP-D binding to CD14 but did not decrease SP-A binding. The SP-A binding to CD14 was completely blocked by a monoclonal antibody that binds to the SP-A neck domain but only partially blocked by an antibody that binds to the SP-A lectin domain. SP-A but not SP-D bound to deglycosylated CD14. SP-D decreased CD14 binding to both smooth and rough LPS, whereas SP-A enhanced CD14 binding to rough LPS and inhibited binding to smooth LPS. SP-A also altered the migration profile of LPS on a sucrose density gradient in the presence of CD14. From these results, we conclude that 1) lung collectins bind CD14, 2) the SP-A neck domain and SP-D lectin domain participate in CD14 binding, 3) SP-A recognizes a peptide component and SP-D recognizes a carbohydrate moiety of CD14, and 4) lung collectins alter LPS/CD14 interactions.

A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides

A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B- and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity forN-acetyl-d-glucosamine orN-acetyl-d-galactosamine. However, the activity of tachylectin-3 was not inhibited by these twoN-acetylhexosamines at 100 mm but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mm. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was fromEscherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.

Rat Mannose-Binding Protein A Binds CD14

ABSTRACT Lipopolysaccharide (LPS) has been known to induce inflammation by interacting with CD14, which serves as a receptor for LPS. Mannose-binding protein (MBP) belongs to the collectin subgroup of the C-type lectin superfamily, along with surfactant proteins SP-A and SP-D. We have recently demonstrated that SP-A modulates LPS-induced cellular responses by interaction with CD14 (H. Sano, H. Sohma, T. Muta, S. Nomura, D. R. Voelker, and Y. Kuroki, J. Immunol. 163:387–395, 2000) and that SP-D also interacts with CD14 (H. Sano, H. Chiba, D. Iwaki, H. Sohma, D. R. Voelker, and Y. Kuroki, J. Biol. Chem. 275:22442–22451, 2000). In this study, we examined whether MBP, a collectin highly homologous to SP-A and SP-D, could bind CD14. Recombinant rat MBP-A bound recombinant human soluble CD14 in a concentration-dependent manner. Its binding was not inhibited in the presence of excess mannose or EDTA. MBP-A bound deglycosylated CD14 treated with N-glycosidase F, neuraminidase, and O-glycosidase, indicating that MBP-A interacts with the peptide portion of CD14. Since LPS was also a ligand for the collectins, we compared the characteristics of binding of MBP-A to LPS with those of binding to CD14. MBP-A bound to lipid A fromSalmonella enterica serovar Minnesota and rough LPS (S. enterica serovar Minnesota Re595 and Escherichia coli J5, Rc), but not to smooth LPS (E. coli O26:B6 and O111:B4). Unlike CD14 binding, EDTA and excess mannose attenuated the binding of MBP-A to rough LPS. From these results, we conclude that CD14 is a novel ligand for MBP-A and that MBP-A utilizes a different mechanism for CD14 recognition from that for LPS.

Horseshoe Crab Factor G: A New Heterodimeric Serine Protease Zymogen Sensitive to (1→3)-β-D-Glucan

Horseshoe crabs (or limulus), as well as other invertebrate animals, do not have an ordinary immune system like mammals. Although they prefer to live in clean oceans, there are still a number of bacteria in their environment. In order to defend themselves from such microorganisms, they have developed a unique and sophisticated defense system in their hemolymph.

Horseshoe Crab Hemocyte- Derived Lectin Recognizing Specific 0-Antigens of Lipopolysaccharides

In the Japanese horseshoe crab, Tachypleus tridentatus, one of the major defense systems is carried by hemolymph that contains granular hemocytes comprising 99% of the total hemocytes. These granular hemocytes are filled with two populations of secretory granules, named large (L) and small (S) granules. These granules selectively store defense molecules, such as clotting serine protease zymogens, a clottable protein coagulogen, protease inhibitors, lectins, and antimicrobial peptides (1–3). The hemocytes are highly sensitive to LPS and these defense molecules stored in both granules are secreted by exocytosis after stimulation with LPS. This response is important for the host defense related to engulfing and killing invading microbes, in addition to preventing the leakage of hemolymph.