Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29–53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.     

A salivary serine protease of the haematophagous reduviid Panstrongylus megistus: sequence characterization,expression pattern and characterization of proteolytic activity

A cDNA encoding a trypsin‐like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin‐like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.     

Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor

The cDNA coding for human monocyte-derived neutrophil-specific chemotactic factor (MDNCF) was cloned from LPS-stimulated human monocyte mRNA. The cDNA sequence codes for a polypeptide consisting of 99 amino acids, including a putative signal sequence. Comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of natural MDNCF shows that the mature functional protein comprises 72 amino acids, beginning with serine at residue 28. The deduced amino acid sequence shows striking similarity to several platelet-derived factors, a v-src-induced protein, a growth-regulated gene product (gro), and an IFN-gamma inducible protein. The availability of the MDNCF cDNA enabled us to use it as a probe to identify inducers of MDNCF mRNA expression in human PBMC. MDNCF mRNA was increased greater than 10-fold within 1 h after stimulation with LPS, IL-1, or TNF, but not by IFN-gamma, IFN-alpha, or IL-2. Furthermore, we also determined that LPS, IL-1, and TNF stimulated the mononuclear cells to produce biologically active MDNCF. This observation may account for the in vivo capacity of IL-1 and TNF to induce netrophil infiltrates.     

Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes

Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2- terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35- kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.     

Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils

Closely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of approximately 29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre- and pro-sequences. The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments. The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization of the full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent.     

The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT‐PCR using degenerate primers based on the N‐terminal amino acid sequence of purified hJHBP and a conserved region near the C‐terminus of other lepidopteran hJHBPs. 5′‐ and 3′‐ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP‐mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS‐PAGE with a M_r of 32 kDa and showed a K_d of 4.5 × 10^?7 M with JH III, both similar to those of native hJHBP.     

Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H (β2-Glycoprotein I)*

Summary Apolipoprotein H, also known as β-₂-glycoprotein I, was purified from human serum, and antiserum produced to denatured apolipoprotein H detected a cDNA clone from a γ gt11 library derived from human liver. This cDNA coded for the complete sequence of the mature protein. The cDNA insert, along with a polymerase chain reaction product which extended the 5′ end of the message, were subcloned and both strands were sequenced. The apolipoprotein H precursor was found to code for 345 amino acids, 326 of which appear in the mature protein. The deduced amino acid sequence of human apolipoprotein H differs from its rat homologue by the presence of a 48-amino acid stretch which is absent from the rat protein. The remainder of the proteins share a greater than 80% similarity. The amino acid sequence of apolipoprotein H consists largely of repeated units approximately 60 amino acids in length. These repeats are comparable to “sushi structures” found in a large number of diverse proteins, including complement components, receptors and regulators of complement activation, serum proteins, membrane-associated adhesion proteins, and other structural and catalytic proteins. Apolipoprotein H was shown to be transcribed by human hepatoma cell lines Hep 3B and Hep G2, and rat liver by detection of mRNA using northern blot analysis. The nucleotide sequence data were deposited in the GenBank Nucleotide Sequence Database, accession number M62839 (April 4, 1991)     

Cloning and expression of a novel juvenile hormone-metabolizing epoxide hydrolase during larval–pupal metamorphosis of the cabbage looper, Trichoplusia ni

A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity. TmEH-1 was 1887 base pairs in lenght with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH III epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH III epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH III as compared to the general EH substrates, cis- and trans-stibene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.     

Cloning and expression of ecto 5'-nucleotidase from the cattle tick Boophilus microplus

Although 5′-nucleotidases are ubiquitous in higher vertebrates, the arthropod enzymes have been little studied. The cDNA sequence of the mature 5′-nucleotidase from the tick Boophilus microplus was therefore determined (Gen Bank accession number: U80634). The enzyme has 39–41% sequence identity with the vertebrate 5′-nucleotidases and contains binuclear metal ion binding sites. There are no significant introns within the coding region of the genomic sequence. Southern blot analysis indicates the presence of multiple related genes encoding 5′-nucleotidases. Recombinant tick 5′-nucleotidase was expressed in both Escherichia coli and in baculovirus-infected insect cells. The E. coli recombinant protein was truncated, inactive and produced in abundance. The enzyme was expressed in baculovirus-infected insect cells as a secreted, soluble, glycosylated and enzymatically active protein. This represents the first successful expression and characterization of enzymatically active recombinant 5′-nucleotidase from any organism. Supplementation of the culture medium with 25 μm zinc resulted in a twofold increase in the activity of the expressed protein. The enzyme was purified to homogeneity. It exists under non-denaturing conditions as a homodimer, with an apparent molecular mass of 135 kDa. The K_m for the hydrolysis of AMP was 0.37 μm and the k_cat= 11.5/s, in agreement with data for the native enzyme.     

cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.     

SECRETION OF PLASMINOGEN ACTIVATOR BY STIMULATED MACROPHAGES

Cultured thioglycollate-stimulated peritoneal macrophages synthesize, accumulate, and continuously release high levels of plasminogen activators for at least 4 days whereas cultures of unstimulated macrophages do not; the higher specific catalytic activity of released vs. cell-associated enzyme suggests that the plasminogen activators are actively secreted. The major macrophage plasminogen activator is a serine protease of mol wt 48,000, and thus resembles the comparable enzyme released by virally transformed fibroblasts. Macrophages release a second plasminogen activator of mol wt 28,000 that is also a serine enzyme. The secretion products released by stimulated and unstimulated macrophages have been compared by SDS-polyacrylamide gel electrophoresis after chemical labeling with ³H-DFP or biosynthetic labeling with ¹⁴C-amino acids. These procedures show that some proteins are formed in both cultures, whereas others are uniquely secreted by each type of macrophage. The serine enzymes released by the two kinds of macrophages differ in specificity and electrophoretic mobility.     

NH2-terminal sequence of macrophage-expressed natural resistance- associated macrophage protein (Nramp) encodes a proline/serine-rich putative Src homology 3-binding domain

The Lsh/Ity/Bcg locus on mouse chromosome 1 regulates macrophage (m phi) priming/activation for antimicrobial activity against intracellular pathogens. A candidate Bcg gene, designated natural resistance-associated m phi protein (Nramp), recently isolated from a pre-B cell cDNA library encodes a polytopic integral membrane protein with structural features common to prokaryotic and eukaryotic transporters. In the present study, an activated m phi cDNA library yielded new Nramp clones that differ in the 5' region from the published pre-B cell-derived clone sequence, resulting in addition of 64 amino acids at the NH2 terminus of the predicted protein. This new domain is rich in proline, serine, and basic amino acids, and includes three protein kinase C phosphorylation sites and a putative Src homology 3 binding domain. RNAs containing this domain are the only form found in the m phi. Hence, the protein encoded by this RNA is the candidate molecule mediating natural resistance to intra-m phi pathogens.     

An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina)

A large and diverse family of serine protease genes was identified in first-instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first-instar larval cDNA, or from third-instar larval salivary glands or cardia, generated using a microscale RT-PCR method, were cloned into a plas-mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue-specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty-nine randomly selected clones from entire first-instar larvae revealed forty-nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first-instar larvae of L. cuprina. DNA sequence analysis of ten randomly-selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first-instar total cDNA and third-instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso-phila melanogaster larval gut cells.