Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.

Peptidoglycans from bacterial cell walls trigger immune responses in insects and mammals. A peptidoglycan recognition protein, PGRP, has been cloned from moths as well as vertebrates and has been shown to participate in peptidoglycan-mediated activation of prophenoloxidase in the silk moth. Here we report that Drosophila expresses 12 PGRP genes, distributed in 8 chromosomal loci on the 3 major chromosomes. By analyzing cDNA clones and genomic databases, we grouped them into two classes: PGRP-SA, SB1, SB2, SC1A, SC1B, SC2, and SD, with short transcripts and short 5-untranslated regions; and PGRP-LA, LB, LC, LD, and LE, with long transcripts and long 5-untranslated regions. The predicted structures indicate that the first group encodes extracellular proteins and the second group, intracellular and membrane-spanning proteins. Most PGRP genes are expressed in all postembryonic stages. Peptidoglycan injections strongly induce five of the genes. Transcripts from the different PGRP genes were found in immune competent organs such as fat body, gut, and hemocytes. We demonstrate that at least PGRP-SA and SC1B can bind peptidoglycan, and a function in immunity is likely for this family.

Insects depend solely upon innate immune responses to survive infection. These responses include the activation of extracellular protease cascades, leading to melanization and clotting, and intracellular signal transduction pathways inducing antimicrobial peptide gene expression. In Drosophila, the IMD pathway is required for antimicrobial gene expression in response to gram-negative bacteria. The exact molecular component(s) from these bacteria that activate the IMD pathway remain controversial. We found that highly purified LPS did not stimulate the IMD pathway. However, lipid A, the active portion of LPS in mammals, activated melanization in the silkworm Bombyx morii. On the other hand, the IMD pathway was remarkably sensitive to polymeric and monomeric gram-negative peptidoglycan. Recognition of peptidoglycan required the stem-peptide sequence specific to gram-negative peptidoglycan and the receptor PGRP-LC. Recognition of monomeric and polymeric peptidoglycan required different PGRP-LC splice isoforms, while lipid A recognition required an unidentified soluble factor in the hemolymph of Bombyx morii.

Innate nonself recognition must rely on common structures of invading microbes. In a differential display screen for up-regulated immune genes in the moth Trichoplusia ni we have found mechanisms for recognition of bacterial cell wall fragments. One bacteria-induced gene encodes a protein that, after expression in the baculovirus system, was shown to be a peptidoglycan recognition protein (PGRP). It binds strongly to Gram-positive bacteria. We have also cloned the corresponding cDNA from mouse and human and shown this gene to be expressed in a variety of organs, notably organs of the immune system-i.e., bone marrow and spleen. In addition, purified recombinant murine PGRP was shown to possess peptidoglycan affinity. From our results and the sequence homology, we conclude that PGRP is a ubiquitous protein involved in innate immunity, conserved from insects to humans.

Three inducible bacteriolytic proteins, designated P7, P9A and P9B, from the hemolymph of immunized pupae of the giant silk moth Hyalophora cecropia have been purified using a two‐step procedure with cation‐exchange chromatography. Purified protein P7 has a molecular weight of 15000 and its amino acid composition shows a great similarity to that of the lysozyme from the wax moth Galleria mellonella. Moreover, heat stability, pH‐rate profile and bacteriolytic specificity also indicate that protein P7 is a lysozyme. The other purified proteins, P9A and P9B, are highly potent against Escherichia coli and some other gram‐negative bacteria. The amino acid compositions of proteins P9A and P9B are very similar, although the contents of glutamic acid and methionine were different. The molecular weights of these very basic proteins are around 7000. The P9 proteins are heat stable; their activities were retained after 30 min incubation at 100°C. Both forms of protein P9 clearly differ from the lysozyme class of enzymes and they may represent a new type of bacteriolytic protein.

1. The steady-state kinetics of the interconversion of CO, and HCO; catalyzed by human carbonic anhydrase C was studied using 'H,O and ' H 2 0 as solvents. The pH-independent parts of the parameters k,,, and K , are 3 -4 times larger in 'H,O than in ,H,O for both directions of the reaction, while the ratios k,,,/K, show much smaller isotope effects. With either C 0 2 or HCO; as substrate the major pH dependence is observed in k,,,, while K , appears independent of pH. The pK, value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2 H 2 0 than in ' H 2 0 .2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhydrase C is approximately 35 faster in 'H20 than in ' H 2 0 . In both solvents the pK, values of the pH-rate profiles are similar to those observed for the C0,-HCO; interconversion.3. It is tentatively proposed that the rate-limiting step at saturating concentrations of C 0 2 or HCO; is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO, and HCO; involves a proton transfer or not.Carbonic anhydrase is a highly efficient catalyst of the reversible interconversion of C 0 2 and HCO,. In a buffered solution not far from neutrality, where Cog-as well as free H + and OH-can be neglected, the stoichiometry of the reaction is CO, + H,O + B e HCO; + BH+, where B and BH' are the basic and acidic buffer components, respectively. Regardless of the specific reaction mechanism, the hydration of C 0 2 must be coupled to the splitting of water, formally into H + and OH-. At some stage of the reaction the OH-ion becomes integrated with C02, while the H + ion ultimately combines with the buffer base. In the reverse reaction OH ~ derived from HCO; must combine with H', originating from the buffer acid, to form H,O. Thus, proton transfers are compulsory ingredients in any mechanism of this reaction.Because of the extremely rapid turnover observed for the enzyme-catalyzed reaction, lo5-lo6 s-' at 25 that H,CO, should be regarded as the substrate species specifically combining with the active site. In effect this means that H + is transported bound to HCO;. It follows from this model that additional proton transfers would have to occur within the enzyme-substrate complex, for example in a concerted reaction as proposed by Kaiser and Lo [3].Arguments against H2C03 as the substrate species combining with the active site have been given by several authors [4-61 pointing out that this would require a second-order rate constant for the binding step exceeding those of diffusion-controlled reactions measured in simpler systems. Alternatively it was suggested that H + is transported between solvent and active site by buffer components acting as proton donors and acceptors. In this case no second-order rate constant involved in the catalytic cycle would have to be greater than lo8-lo9 M-' s -l , and it is not necessary to invoke novel phenomena such as surface diffusion [2,7] to rationalize the e...

Recent studies of peptidoglycan recognition protein (PGRP) have shown that 2 of the 13 Drosophila PGRP genes encode proteins that function as receptors mediating immune responses to bacteria. We show here that another member, PGRP-SC1B, has a totally different function because it has enzymatic activity and thereby can degrade peptidoglycan. A mass spectrometric analysis of the cleavage products demonstrates that the enzyme hydrolyzes the lactylamide bond between the glycan strand and the cross-linking peptides. This result assigns the protein as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), and the corresponding gene is thus the first of this class to be described from a eukaryotic organism. Mutant forms of PGRP-SC1B lacking a potential zinc ligand are enzymatically inactive but retain their peptidoglycan affinity. The immunostimulatory properties of PGRP-SC1B-degraded peptidoglycan are much reduced. This is in striking contrast to lysozyme-digested peptidoglycan, which retains most of its elicitor activity. This points toward a scavenger function for PGRP-SC1B. Furthermore, a sequence homology comparison with phage T7 lysozyme, also an N-acetylmuramoyl-L-alanine amidase, shows that as many as six of the Drosophila PGRPs could belong to this class of proteins.

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