Biomedical subjects

Haobo Jiang

Publications and source records attributed to Haobo Jiang.

25 records · Page 2Linked to original sources

PubMed2003

Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases.

Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses. We have isolated an immune-responsive serpin from the tobacco hornworm, Manduca sexta. This inhibitor, M. sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO). Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization. M. sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5-12 microg/ml and increases to 30-75 microg/ml after a microbial challenge. Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M. sexta. PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.

Animals↗

PubMed2003

Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships.

Serine proteases (SPs) and serine protease homologs (SPHs) constitute the second largest family of genes in the Drosophila melanogaster genome. Eighty-four SPs comprise less than 300 amino acid residues, and a significant portion of them are probably digestive enzymes. Some larger SPs may contain one or more regions important for protein-protein interactions, including clip domains, low-density lipoprotein receptor class A repeats, and scavenger receptor cysteine-rich domains. We identified 37 clusters of SP or SPH genes, which probably evolved from relatively recent gene duplication and sequence divergence. A majority of the SPs may be trypsin-like and activated by cleavage after a specific arginine or lysine residue. Among the 147 SPs and 57 SPHs studied, 24 SPs and 13 SPHs contain at least one regulatory clip domain. A multiple sequence alignment of the clip domains provided further information on structural conservation of these regulatory modules. Detailed sequence comparison led to an improved classification system for SPs containing clip domains. These analyses have established a framework of information about evolutionary relationships among the Drosophila SPs and SPHs, which may facilitate research on these proteins as well as homologous molecules from other invertebrate species.

Amino Acid Sequence↗

PubMed2003

Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm, Manduca sexta.

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.

Amino Acid Sequence↗

PubMed2003

Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs.

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.

Amidohydrolases↗

PubMed2003

Expression and in vitro activation of Manduca sexta prophenoloxidase-activating proteinase-2 precursor (proPAP-2) from baculovirus-infected insect cells.

Prophenoloxidase activation is a component of the immune system in insects and crustaceans. We recently purified and cloned a new prophenoloxidase-activating proteinase (PAP-2) from hemolymph of the tobacco hornworm Manduca sexta [J. Biol. Chem. 278, 3552-3561]. As the terminal component of a putative serine proteinase cascade, this enzyme activates prophenoloxidase (proPO) via limited proteolysis. To purify and study the activating proteinase for PAP-2 from this insect, we expressed the zymogen of PAP-2 (proPAP-2) in insect cells infected by a recombinant baculovirus that harbors the cDNA. To facilitate the purification of proPAP-2, we modified a commercial vector (pFastBac1) by inserting a synthetic DNA fragment encoding a hexahistidine sequence, allowing fusion of the affinity tag to the carboxyl terminus of a protein. After Spodoptera frugiperda Sf21 cells were infected by the virus, recombinant proPAP-2 was efficiently secreted into the media at a concentration of 5.9 microg/ml under the optimal conditions. After ammonium sulfate precipitation, the proenzyme was purified to near homogeneity by affinity chromatography on Ni(2+)-NTA agarose. Western blot analysis indicated that the recombinant proPAP-2 has a mobility slightly lower than that of the zymogen from M. sexta hemolymph. The molecular mass and isoelectric point of proPAP-2 were determined to be 47,573+/-11Da and 6.6, respectively. After the purified proenzyme was added to hemolymph from induced M. sexta larvae, it was rapidly activated by an unknown proteinase in the presence of peptidoglycan.

Amidohydrolases↗

PubMed2002

A serpin mutant links Toll activation to melanization in the host defence of Drosophila.

A prominent response during the Drosophila host defence is the induction of proteolytic cascades, some of which lead to localized melanization of pathogen surfaces, while others activate one of the major players in the systemic antimicrobial response, the Toll pathway. Despite the fact that gain-of-function mutations in the Toll receptor gene result in melanization, a clear link between Toll activation and the melanization reaction has not been firmly established. Here, we present evidence for the coordination of hemolymph-borne melanization with activation of the Toll pathway in the Drosophila host defence. The melanization reaction requires Toll pathway activation and depends on the removal of the Drosophila serine protease inhibitor Serpin27A. Flies deficient for this serpin exhibit spontaneous melanization in larvae and adults. Microbial challenge induces its removal from the hemolymph through Toll-dependent transcription of an acute phase immune reaction component.

Animals↗

PubMed2002

Prophenoloxidase-activating proteinase-2 from hemolymph of Manduca sexta. A bacteria-inducible serine proteinase containing two clip domains.

Proteolytic activation of prophenoloxidase in insects is a component of the host defense system against invading pathogens and parasites. We have purified from hemolymph of the tobacco hornworm, Manduca sexta, a new serine proteinase that cleaves prophenoloxidase. This enzyme, designated prophenoloxidase-activating proteinase-2 (PAP-2), differs from another PAP, previously isolated from integuments of the same insect (PAP-1). PAP-2 contains two clip domains at its amino terminus and a catalytic domain at its carboxyl terminus, whereas PAP-1 has only one clip domain. Purified PAP-2 cleaved prophenoloxidase at Arg(51) but yielded a product that has little phenoloxidase activity. However, in the presence of two serine proteinase homologs, active phenoloxidase was generated at a much higher level, and it formed covalently linked, high molecular weight oligomers. The serine proteinase homologs associate with a bacteria-binding lectin in M. sexta hemolymph, indicating that they may be important for ensuring that the activation of prophenoloxidase occurs only in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in naive larval fat body or hemocytes, but it became abundant in these tissues after the insects were injected with bacteria.

Amino Acid Sequence↗