Haobo Jiang

Oxidative conjugation of catechols with proteins in insect skeletal systems

Abstract Cuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenylethanol. C–N and C–O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol–amino acid adducts during sclerotization.

Characterization and Functional Analysis of 12 Naturally Occurring Reactive Site Variants of Serpin-1 from Manduca sexta

Serpin gene-1 from the tobacco hornworm, Manduca sexta, encodes, through alternative exon usage, 12 reactive site variants (Jiang, H., Wang, Y. and Kanost, M. R., (1994) J. Biol. Chem. 269, 55-58; Jiang, H., Wang, Y., Huang, Y., Mulnix, A. B., Kadel, J., Cole, K., and Kanost, M. R. (1996) J. Biol. Chem. 271, 28017-28023). These 43-kDa proteins differ from each other only in their COOH-terminal 39-46 residues, which include the reactive site. To test the hypothesis that these proteins are proteinase inhibitors of diverse selectivities and to begin to elucidate their physiological functions, we expressed the 12 serpin-1 variants in Escherichia coli. Seven of the variants inhibited mammalian serine proteinases, with association rate constants comparable with those of human serpins. Serpin-1A, with a P1 Arg residue, inhibited both trypsin and plasmin. Serpin-1B (P1 Ala) and serpin-1F (P1 Val) inhibited porcine pancreatic elastase and human neutrophil elastase. Serpin-1H, −1K, and −1Z, all with a Tyr residue at the P1 position, inhibited chymotrypsin and cathepsin G. Serpin-1I (P1 Leu) inhibited both elastase and chymotrypsin. Nine of the serpin variants were active as inhibitors of microbial serine proteinases, including subtilisin Carlsberg, proteinase K, and two proteinases secreted by an entomopathogenic fungus, Metarhizium anisopliae. In addition, one of the serpin variants, serpin-1J, strongly inhibited activation of M. sexta hemolymph phenoloxidase, a pathway involving a serine proteinase cascade. This pathway is a component of the defensive response of insects to microbial infection. These results suggest that the products of M. sexta serpin gene-1 may be important in regulating both exogenous and endogenous serine proteinases in hemolymph.

Subunit Composition of Pro-phenol Oxidase from Manduca sexta: Molecular Cloning of Subunit ProPO-p1

Phenol oxidase (PO) is known to play an important role in defense mechanisms in insect immunity. It is present as a zymogen in insect hemolymph, and can be activated by a specific proteolytic reaction that is stimulated by microbial cell wall components. The pro-phenol oxidase (pro-PO) purified from the larval hemolymph of Manduca sexta contains two polypeptides in equal amounts as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cDNA for one of the polypeptides, now designated proPO-p2, has been isolated (Hall et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 7764-7768). We purified pro-PO from plasma of M. sexta and characterized its subunit composition. A cDNA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA library. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bombyx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immunofluorescence labelling and in situ hybridization showed that the pro-PO is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multimeric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a detergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of distinct mobility (fast and slow forms). Both forms contain proPO-p1 and proPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indicate that there are two pro-PO genes in M. sexta, which are coordinately expressed in oenocytoids, and whose products form predominantly heterodimers in plasma.

Organization of Serpin Gene-1 from Manduca sexta EVOLUTION OF A FAMILY OF ALTERNATE EXONS ENCODING THE REACTIVE SITE LOOP

Manduca sexta serpin gene-1 encodes a family of serpins whose amino acid sequences are identical in their amino-terminal 336 residues but variable in their carboxyl-terminal 39-46 residues, which includes the reactive site loop (Jiang, H., Wang, Y., and Kanost, M. R. (1994) J. Biol. Chem. 269, 55-58). Here, we report the genes complete nucleotide sequence and exon-intron structure. A unique characteristic of this gene is its exon 9, which is present in 12 alternate forms between exons 8 and 10. Isolation and characterization of cDNA clones containing exons 9C, 9H, and 9I, which were not found previously, indicate that all 12 alternate forms of exon 9 can be utilized to generate 12 different serpins. The splicing pathway apparently allows inclusion of only one exon 9 per molecule of mature serpin-1 mRNA. Analysis of exon-intron border sequences reveals unique features that may be involved in regulation of RNA splicing. The exon 9 region has apparently evolved through rounds of exon duplication and sequence divergence. The exons near the center of the region may have evolved recently, whereas the outermost exons are the most ancient. Exons 9G and 9H were duplicated as a pair from exons 9E and 9F, an event that may have occurred more than once in the history of this gene.

Biological activity of Manduca sexta paralytic and plasmatocyte spreading peptide and primary structure of its hemolymph precursor

A family of hemolymph peptides was previously identified in several lepidopteran insects, which exhibited multiple biological activities including rapid paralysis, blockage of growth and development, or stimulation of plasmatocyte spreading and aggregation. We synthesized Manduca sexta paralytic peptide 1 (PP1) and found that after it was injected into larvae, bleeding from wounds was dramatically reduced. PP1 also stimulated spreading and aggregation behavior of M. sexta plasmatocytes in vitro. Stimulation of plasmatocyte aggregation and adherence to the body wall may explain a decrease observed in the number of circulating plasmatocytes after injection of PP1. Such aggregates might rapidly form plugs in wounds to prevent bleeding. We cloned a cDNA for a Manduca paralytic peptide precursor, using polymerase chain reactions and cDNA library screening. The active 23-residue PP2 peptide encoded by this clone is at the carboxyl-terminal end of a precursor protein predicted to be 107 amino acid residues long after cleavage of a secretion signal peptide. Active PP2 was produced by processing of recombinant proPP2 by bovine factor Xa. A single proPP2 mRNA was present in fat body but not in hemocytes. The level of this mRNA was not affected by injection of bacteria into larvae. We produced recombinant proPP2 in Escherichia coli and used this protein to produce an antiserum. The antiserum detected proPP2 in plasma and was used to observe rapid proteolytic processing of proPP2 after hemolymph collection.

The structure of active serpin 1K from Manduca sexta

BACKGROUNDnThe reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding.nnnRESULTSnThe 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin.nnnCONCLUSIONSnSerpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.

Primary structure of ribosomal proteins S3 and S7 from Manduca sexta.

We have isolated from Manduca sexta full‐length cDNAs encoding proteins homologous to human ribosomal proteins S3 and S7. These are the first ribosomal protein sequences obtained from non‐Dipteran insects. M, sexta ribosomal protein S3 has a molecular mass of 26,715 Da. Ribosomal protein S7 has a mass 21,870 Da. Both are basic proteins, with abundant Lys and Arg residues that may interact with ribosomal RNA in the ribosome. Southern blot hybridization suggests the presence of single genes for both ribosomal proteins in the M. sexta genome. Alignments with other S3 and S7 sequences available in the database indicate regions of the ribosomal proteins that have been the most highly conserved in evolution and may point to important functional regions in the proteins. Ribosomal protein S3 appears to be more highly conserved than ribosomal protein S7. This may be due to greater constraints on the structure of S3 because of its dual functions in translation as a ribosomal protein and in DNA repair in the nucleus.

Hemolymph Proteinases in Immune Responses of Manduca sexta

Extracellular signal transduction pathways in blood are often composed of cascades of serine proteinases to amplify rapidly a response to wounding or infection. The vertebrate blood coagulation and complement pathways are the most fully characterized of such proteinase cascades. Serine proteinases also play important roles in defensive responses in hemolymph of arthropods. The hemolymph coagulation system of horseshoe crabs has been very nicely elucidated to function as a complex and intricately regulated proteinase cascade for protection against microbial infection (Kawabata et al., 1996).

Molecular cloning of cDNAs for two pro-phenol oxidase subunits from the malaria vector, Anopheles gambiae

Phenol oxidase exists in insect hemolymph as a zymogen, pro-phenol oxidase (pro-PO), which is activated by specific proteolysis in response to infection or wounding. Phenol oxidase catalyses the synthesis of quinones that polymerize to form melanin deposits, which encapsulate parasites and help to seal wounds. Antibodies to pro-PO from Manduca sexta bound to 76, 72, and 71 kDa polypeptide bands from hemolymph of Anopheles gambiae larvae. This antiserum was used to screen a cDNA library from A. gambiae fourth-instar larvae. Full-length clones were isolated for two different pro-POs, designated A. gambiae proPO-p1 and proPO-p2, which are 67% identical in nucleotide sequence and 66% identical in deduced amino acid sequence. The A. gambiae pro-PO sequences are more similar to pro-PO from Drosophila melanogaster than to lepidopteran or crustacean pro-PO sequences in the GenBank database. Like the other arthropod pro-POs, the A. gambiae pro-PO sequences lack a signal peptide and have two conserved regions predicted to bind two copper atoms in the active site of the enzyme. The availability of these pro-PO cDNAs should be useful in examining the biochemical differences between A. gambiae strains that are refractory or susceptible to Plasmodium infection, and differ in their ability to encapsulate the parasites.

A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta.

Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.