The role of HiTI, a serine protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases

Blood-sucking arthropods are vectors responsible for the transmission of several pathogens and parasites to vertebrate animals. The horn fly Haematobia irritans irritans (Diptera: Muscidae) and the tick Boophilus microplus are important hematophagous ectoparasites that cause losses in cattle production. A serine protease inhibitor from a thorax extract of the fly H. irritans irritans (HiTI) was previously isolated, characterized and cloned. In the present study we described the expression, purification, and characterization of the recombinant HiTI (rHiTI) and its possible role in the control of different endogenous and bacterial proteases. rHiTI was successfully expressed using the pPIC9 expression vector with a yield of 4.2 mg/L of active rHiTI. The recombinant HiTI purified by affinity chromatography on trypsin-Sepharose had a molecular mass of 6.53 kDa as determined by LS-ESI mass spectrometry and inhibition constants (Kis) similar to those of native HiTI for bovine trypsin and human neutrophil elastase of 0.4 and 1.0 nM, respectively. Purified rHiTI also showed inhibitory activity against the trypsin-like enzyme of H. i. irritans using its possible natural substrates, fibrinogen and hemoglobin; and also inhibited the OmpT endoprotease of Escherichia coli using fluorogenic substrates. The present results confirm that HiTI may play a role in the control of fly endogenous proteases but also suggest a role in the inhibition of pathogen proteases.     

Identification and characterization of a cDNA encoding the acetylcholinesterase of Haematobia irritans (L.) (Diptera: Muscidae).

A 2217-nucleotide cDNA presumptively encoding acetylcholinesterase (AChE) of the horn fly, Haematobia irritans (L.) was sequenced. The open reading frame (ORF) encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 95% identity and 98% similarity to the AChE of Musca domestica (L.). Structural features characteristic of the M. domestica and Drosophila melanogaster AChEs are conserved in the H. irritans AChE. The M. domestica and D. melanogaster AChEs are target sites for organophosphate inhibition as previously shown (Walsh et al. 2001. Biochem. J. 359: 175-181, Kozaki et al. 2002. Appl. Entomol. Zool. 37: 213-218), suggesting that this H. irritans AChE2 may be the target site for organophosphate.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen.

The second major allergen of Chamaecyparis obtusa (Japanese cypress) pollen, Cha o 2, has been purified and its cDNA cloned. Of patients with pollinosis caused by C. obtusa, 82.5% produce IgE antibodies which react with purified Cha o 2. The purified protein has a molecular mass of 46 kDa and its 12 N-terminal amino acid sequence displays a high homology with that of Cry j 2, the second major allergen of Cryptomeria japonica pollen. cDNA clones coding for Cha o 2 have been isolated using Cry j 2 cDNA as a probe. Cha o 2 cDNA clones were sequenced and found to code a putative 50-residue signal sequence and a 464-residue mature protein with a molecular weight of 50 kDa. Two possible N-linked glycosylation sites were found in the sequence. The deduced amino acid sequence of Cha o 2 shows 74.3% identity with that of Cry j 2. In its primary structure, Cha o 2 shows significant identity with those of the polygalacturonases of avocado, tomato, and maize as well as Cry j 2.     

Molecular cloning of a putative agglutination/immobilization antigen located on the surface of a novel agglutination/immobilization serotype of Cryptocaryon irritans

A surface agglutination/immobilization antigen was purified from the novel agglutination/immobilization serotype (serotype G37) of the ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes. Serum from fish immunized with C. irritans theronts had agglutination/immobilization activity against theronts in vitro. However, fish and rabbit antisera raised against serotype G32 (reported previously) caused little agglutination/immobilization of serotype G37 theronts. Immunological analysis indicated that the 37 kDa theront surface membrane protein may be the agglutination/immobilization antigen of this serotype. The full-length 37 kDa antigen cDNA contained 1171 base pairs, encoding a 331-amino acid protein with hydrophobic N- and C-termini, which are characteristically found in proteins containing a C-terminal glycosylphosphatidylinositol anchor. In addition, the genetically characterized nucleotide sequences of the first internal transcribed spacer region of ribosomal DNA of these 2 serotypes were compared. The internal transcribed spacer rDNA sequence of serotype G32 was identical to that of isolates from Pingtung, Taiwan, and from the USA. On the other hand, the sequences of serotype G37 were not identical to those of any C. irritans isolate.     

Isolation and characterization of a trypsin-like enzyme from the buffalo fly, Haematobia irritans exigua

Abstract. The incorporation of soybean trypsin inhibitor (SBTI) into the diet of the buffalo fly, Haematobia irritans exigua (De Meijere), results in increased mortality and reduced fecundity. A trypsin-like enzyme which binds to SBTI was isolated by affinity chromatography on a Sepharose-SBTI column followed by ion-exchange chromatography. The enzyme was inhibited by benzamidine, phenylmethylsulfonyl fluoride, ovomucoid, leupeptin and a-2 macroglobulin. The enzyme was not inhibited by EDTA or p-chloromecuribenzoic acid and had a broad pH optimum of pH 7–9. Vaccination of sheep produced antibodies specific for the trypsin-like enzyme which inhibited enzyme activity in vitro but did not affect the survival of flies maintained in in vitro culture.     

Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica.

S-adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5'- and 3'-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity.     

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen

The second major allergen of Juniperus ashei (mountain cedar) pollen, Jun a 2, has been purified and its cDNA cloned. The purified protein has a molecular mass of 43 kDa and its N-terminal 9-residue amino acid sequence is highly homologous to those of Cry j 2 and Cha o 2, the second major allergen of Cryptomeria japonica and Chamaecyparis obtusa pollen, respectively. cDNA clones encoding Jun a 2 were isolated after PCR based amplification, and their nucleotide sequences were determined. The cDNA contains an open reading frame of 507 amino acid residues, and encodes a putative 54-residue signal sequence and a 453-residue intermediate, which releases a C-terminal fragment upon maturation. Three possible N-linked glycosylation sites and 20 cystein-residues are found in the deduced amino acid sequence. The amino acid sequence of Jun a 2 shows 70.7 and 82.0% identity with those of Cry j 2 and Cha o 2, respectively. Immunological observations that IgE antibodies in sera of Japanese pollinosis patients bind not only to Cry j 2 and Cha o 2 but also to Jun a 2 strongly suggest that Jun a 2 is an allergen of mountain cedar pollen, and that allergenic epitopes of these three allergens are similar.     

Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva.

Thrombostasin (TS), a novel protein found in the saliva of Haematobia irritans (horn fly), was purified by high-performance liquid chromatography from the saliva of field-collected insects. This protein, which inhibits thrombin, accounts for anti-clotting activity in horn fly saliva [J. Med. Entomol. 37 (2000) 416] and is the first purified anti-hemostatic factor described from the Stomoxyinae, a large group of blood-feeding insects that are major pests of livestock world-wide. The purified TS had an apparent molecular weight of 16.7 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed two isoelectric groups with isoelectric points (pIs) of approximately 4.6 and 4.8. Mass spectroscopy analysis, however, resulted in at least three major isoforms that range in mass from 9213 to 9274 Da. A 243-bp coding sequence was obtained from cDNA by using a degenerate primer deduced from the N-terminal sequence of the purified TS. The conceptual translation of the 243-bp sequence showed that the 81-amino-acid peptide, whose first 30 amino acids match those of the N-terminal sequence, had a predicted mass of 9213 Da with pI 4.14. A full-length TS cDNA was generated by rapid amplification of cDNA ends of the 5' and sequential polymerase chain reaction (PCR) amplification. It contained a 5'-end 12-bp segment preceding the putative ATG start codon, followed by a 54-bp sequence corresponding to a secretory signal and an additional 228-bp coding sequence preceding residues revealed by N-terminal sequencing of purified TS. The fidelity of the PCR-generated TS sequence was confirmed in genomic DNA and by biological activity of recombinant TS produced in a baculovirus expression system. Database comparisons revealed no homology between TS and other known molecules. Because of the paucity of other anti-hemostatic factors in horn fly saliva, TS may play a critical role in maintenance of the ectoparasitic lifestyle of horn flies.     

Identification of a basic glycoprotein induced by ethylene in primary leaves of azuki bean as a cationic peroxidase.

Ethylene causes the accumulation of seven different proteins (each designated AZxx according to its molecular mass, xx in kD) in excised primary leaves of azuki bean (Vigna angularis) (F. Ishige, H. Mori, K. Yamazaki, H. Imaseki [1991] Plant Cell Physiol 32: 681-690). A complementary DNA encoding an ethylene-induced basic glycoprotein, AZ42, from azuki bean was cloned and its complete nucleotide sequence was determined. Characterization of the cDNA was accomplished by monitoring expression of an immunoreactive protein in Escherichia coli that harbored the cDNA and by the identification of a partial amino acid sequence that was the same as that determined from the purified protein. An open reading frame (1071 base pairs) in the cDNA encoded a protein of 357 amino acids with a molecular mass of 39.3 kD. The amino acid sequence contained three regions that are highly conserved among peroxidases from eight different plants. Purified AZ42 exhibited peroxidase activity. The basic glycoprotein induced by ethylene was identified as a cationic isozyme of peroxidase. The corresponding mRNA was not present in leaves that had not been treated with ethylene, but it appeared after 1 h of treatment with ethylene and its level increased for the next 15 h. Accumulation of the mRNA was also induced after wounding or treatment with salicylate. The wound-induced increase in the level of the mRNA was suppressed by 2,5-norbornadiene, but the salicylate-induced increase was not.     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

Primary structure of rat liver 5'-nucleotidase deduced from the cDNA. Presence of the COOH-terminal hydrophobic domain for possible post-translational modification by glycophospholipid

Rat liver 5'-nucleotidase was purified from a crude microsomal fraction, and its molecular mass was estimated to be 73 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein was subjected to cleavage with CNBr or lysyl endopeptidase, and the resulting 21 peptides as well as the NH2 terminus of the native protein were sequenced by Edman degradation. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for 5'-nucleotidase, lambda cNTP6 and lambda cNT34. The 3.2-kilobase cDNA insert of lambda cNTP6 contains an open reading frame that encodes a 576-residue polypeptide with a calculated size of 63,965 Da, which is in reasonable agreement with that of 5'-nucleotidase (62 kDa) immunoprecipitated from cell-free translation products. The NH2-terminal 28 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. The predicted structure contains all the other peptide sequences determined by Edman degradation. Five potential N-linked glycosylation sites are found in the molecule, accounting for the difference in mass between the precursor and mature forms. Another characteristic feature is that the primary structure contains a highly hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. In fact, labeling experiments of rat hepatocytes demonstrated that 3H-labeled compounds such as ethanolamine, myo-inositol, and palmitic acid, components of the glycolipid anchor, were incorporated into 5'-nucleotidase. Phosphatidylinositol-specific phospholipase C released 5'-nucleotidase from the cell surface, and the released protein no longer contained the radioactivity of [3H]palmitic acid incorporated.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs

Inosine-5'-monophosphate dehydrogenase, a key enzyme in the regulation of guanine nucleotide biosynthesis, was purified to homogeneity; and a polyclonal antibody directed against the purified protein was used to isolate human and Chinese hamster IMP dehydrogenase cDNA clones. These clones were sequenced and found to contain an open reading frame of a protein containing 514 amino acids. A sequence of 35 amino acids obtained by analysis of the purified protein is identical to a segment of the protein sequence deduced from the IMP dehydrogenase cDNA. The molecular mass of the deduced protein is 56 kDa, which is the observed molecular mass of the purified protein and of the immunoprecipitated in vitro translation product. Comparison of the protein sequences deduced from the human and Chinese hamster cDNA clones indicates only eight amino acid differences, suggesting that IMP dehydrogenase is a highly conserved protein.     

Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences.