George A. Riding

Characterization of a Major Peritrophic Membrane Protein, Peritrophin-44, from the Larvae of Lucilia cuprina cDNA AND DEDUCED AMINO ACID SEQUENCES

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Isolation from the cattle tick, Boophilus microplus, of antigenic material capable of eliciting a protective immunological response in the bovine host

Abstract Willadsen P. , Mckenna R. V. and Riding G. A. 1988. Isolation from the cattle tick, Boophilus microplus, of antigenic material capable of eliciting a protective immunological response in the bovine host. International Journal for Parasitology18: 183–189. Vaccination of cattle with material derived from semi-engorged female Boophilus microplus produces an immunity to the parasite different from and often more efficacious than naturally acquired immunity. Practical exploitation of this would require the isolation and identification of the antigens responsible. It has been shown that protective antigens can be isolated from crude membrane preparations by detergent extraction, gel permeation chromatography and preparative isoelectric tocussing. Appropriate combination of these techniques allows a high degree of purification to be achieved.

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.

TaNAC69 from the NAC superfamily of transcription factors is up-regulated by abiotic stresses in wheat and recognises two consensus DNA-binding sequences

NAC proteins are one of the largest families of plant transcription factors and have recently been implicated in diverse physiological processes. To elucidate their role in gene regulation, we determined the DNA-binding specificity of a drought- and cold-inducible NAC protein, TaNAC69 from wheat, and analysed its homologues from other species. Two consensus DNA-binding sequences (spanning 23-24 bp) of TaNAC69 were identified through binding site selection and both consisted of two half sites. Comprehensive data on the DNA-binding specificity of TaNAC69 were generated through extensive base substitution mutagenesis. TaNAC69 and its homologue in Arabidopsis, NAP, sharing 75% sequence identity in the NAC domain, exhibited similar DNA-binding specificity. TaNAC69 was able to homodimerise through its NAC domain. The NAC domain consists of five conserved subdomains. Subdomain mutation showed that a loss or reduction in TaNAC69 dimerisation capacity was accompanied with abolition or decrease in its DNA-binding activity. These data suggest that all subdomains are necessary to maintain a functional NAC domain structure required for interaction with DNA and dimerisation.

Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA

Two chymotrypsin‐like proteases were purified from the secretory and excretory material of first‐instar larvae of Lucilia cuprina. The hydrolysis of N‐succinyl‐L‐phenylalanine‐nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor‐Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease {Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis‐causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

CDNA AND DEDUCED AMINO ACID SEQUENCES OF A PERITROPHIC MEMBRANE GLYCOPROTEIN, 'PERITROPHIN-48', FROM THE LARVAE OF LUCILIA CUPRINA

The gut of most insects is lined with a semi-permeable peritrophic membrane (or peritrophic matrix) composed of chitin, proteoglycans and proteins. Despite the probable importance of the peritrophic membrane in facilitating the digestive process and protecting insects from invasion by micro-organisms and parasites, there has been little characterization of the specific components and their interactions within this acellular structure. Here we report the characterization of an integral peritrophic membrane glycoprotein, peritrophin-48, from the larvae of the fly Lucilia cuprina, a primary agent of cutaneous myiasis in sheep. Peritrophin-48 was purified from peritrophic membrane obtained by larval culture and its location within the peritrophic membrane determined by immuno-fluorescence and immuno-gold localizations. The cDNA coding for peritrophin-48 was cloned and sequenced. The deduced amino acid sequence codes for a protein of 375 amino acids containing an amino-terminal signal sequence followed by five similar, but non-identical domains, each approximately 65-70 amino acids in length and characterised by a specific register of six cysteines. The deduced amino acid sequence shows significant similarity to two other peritrophic membrane proteins, peritrophin-95 and peritrophin-44, from the same species. A reverse transcriptase-PCR approach indicated that there are several highly related peritrophin-48 genes expressed in each individual. Reverse transcriptase-PCR also demonstrated the expression of peritrophin-48 in all three larval instars and adults but not pupae or eggs. Peritrophin-48 was expressed only by the cardia and by the larval midgut. A simple structural model of a basic unit of a type 2 peritrophic membrane is presented.

Vaccination of cattle against the Boophilus microplus using a mucin‐like membrane glycoprotein

An antigen, BMA7, which induced partial immunity against tick infestation has been isolated from Boophilus microplus using two different protein fractionation protocols, accompanied by vaccination and parasite challenge trials. The antigen is a 63 kDa glycoprotein isolated from semi‐engorged adult female ticks. Though significant, the induced immunity is less striking than that previously reported for antigen Bm86 from the same parasite. However, co‐vaccination with Bm86 and BMA7 can enhance immunity over that seen with a commercial vaccine based on Bm86 alone. Limited peptide sequence information shows significant variation in the BMA7 protein occurs. The antigen has approximately 36 kDa of glycosylation, in both N‐linked and O‐linked oligosaccharides. There is evidence that both polypeptide and oligosaccharide are antigenic, but the chemical nature of the protective antigenic sites is not clear. There is little or no immunological response to the antigen during natural infestation with parasites, suggesting the antigen is ‘concealed’ and protective immunity dependent on artificial vaccination. The antigen has some similarities with the vertebrate mucins. It is widely distributed in tick tissues and membrane bound but its function is currently unknown.

Carboxydipeptidase from Boophilus microplus: A “concealed” antigen with similarity to angiotensin-converting enzyme

A protein, Bm91, which was first identified as a protective vaccine antigen from the tick Boophilus microplus, has regions of very strong amino acid sequence similarity to mammalian carboxydipeptidases or angiotensin converting enzymes (ACE; E.C. 3.4.15.1). This protein is now shown to share many biochemical and enzymatic properties with mammalian carboxydipeptidases. It is enzymatically active in a conventional assay for ACE using hippuryl-Gly-Gly as substrate. The hydrolysis of the C-terminal nonapeptide of the insulin B chain proceeds by sequential removal of carboxy-terminal dipeptides. The similarities extend to the dependence of activity on pH and added salt. Bm91 is inhibited by two well-characterized inhibitors of the mammalian enzymes, the drug Captopril and a nonapeptide, and the inhibition occurs in similar concentration ranges to those effective with the mammalian enzymes. However, the natural substrates of the tick enzyme are unknown. Angiotensin I itself is a poor substrate and the enzymes natural substrates are likely to be one or more of the pharmacologically active peptides occurring in insects and arthropods.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4+ T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-γ, and tumor necrosis factor-α, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Potential for recombinant Babesia bovis antigens to protect against a highly virulent isolate

Two antigens from Babesia bovis,12D3 and 11C5, were expressed and purified as recombinant proteins in Escherichia coli and used to vaccinate groups of six Babesia‐susceptible cattle. These were subsequently challenged with a highly virulent strain of B. bovis. All cattle showed symptoms of disease and most required treatment. Cattle vaccination groups receiving either 12D3 or 11C5 or a combination of both, reduced parasitaemia by approximately fourfold and a number of individual animals appeared to control the parasite infection. Control of parasites correlated with high monocyte numbers late in infection. The results thus confirm the potential usefulness of both antigens but also demonstrate the limitations of current formulations.