| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2004 as doi:10.1096/fj.03-1154fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cellular Regulation and Signaling Division, School of Biological Sciences, University of Liverpool, Liverpool, UK;
* Ciphergen Biosystems Inc., Fremont, California, USA, and
Novartis Animal Health Centre de Recherche Santé Animale, St.-Aubin, Switzerland
1 Correspondence: Cellular Regulation and Signaling Division, School of Biological Sciences, University of Liverpool, Biosciences Building, Crown St., Liverpool, L69 7ZB, UK. E-mail: reeshh{at}liv.ac.uk
SPECIFIC AIMS
In the course of investigating the innate immunity of ticks during blood-feeding, a new type of antimicrobial protein was identified, named hebraein, with multiple histidines and a novel secondary structure composed of four to six 
-helices. Our aims were to study the pH-dependency of hebraein’s antimicrobial activities, its recombinant expression, and its differential expression during feeding.
PRINCIPAL FINDINGS
1. Cloning of hebraein cDNA and recombinant expression
Our hebraein cDNA clone contained an insert of 0.7 kb encoding a small predicted protein with 6 cysteine residues and multiple histidine residues in its carboxyl-terminal region (GenBank accession number AY437139). Conceptual translation of the cDNA sequence suggests that hebraein (102 amino acids) is produced as a pro-protein consisting of 123 amino acid residues with 9 histidine residues present as 8 His-X repeats (NH2......106HDHDHDHGHGHGHDHDPH-COOH) at its carboxyl terminus. This latter feature suggested that hebraein may have antimicrobial activity because there are some antimicrobial peptides that are rich in specific amino acids, such as histidine or tryptophan. As no similarity was found to any known protein in existing databases at the time of cloning, hebraein was clearly a novel protein and warranted further study. Native hebraein was purified from the hemolymph of fed female ticks and its amino-terminal amino acid sequence was determined. This matched the amino acid sequence deduced from the cDNA at positions 22-38. Its molecular mass (11,427.1) determined by MALDI-TOF analysis also matched well with the predicted molecular mass (11, 434 Da) of the mature protein and suggests that all six cysteines form disulfide bridges. Recombinant hebraein and a histidine-deficient mutant, lacking the eight HX repeats, were expressed in High Five cells and purified.
2. Secondary structure prediction
Secondary structure prediction strongly suggests that hebraein is a protein with multiple -helixes (4-6 -helixes) distributed evenly throughout its length. Such an all-helical structure is quite different from other known antimicrobial proteins/peptides that possess either a single -helix or a cystine-stabilized ß-sheet structure.
3. Antimicrobial activity and its pH dependency
Native hebraein purified from the hemolymph of fed female ticks and the two recombinant hebraeins expressed in insect cells were assayed for their antimicrobial activities under different pH conditions. The antimicrobial activities (minimal inhibitory concentration, MIC) of these three forms of hebraein against S. aureus Oxford, E. coli OP 50, C. albicans SC 5314, and C. glabrato ATCC 2001 were determined. The antimicrobial activities of the histidine-rich, wild-type hebraein samples are substantially greater at pH 6.2 than at pH 7.6. By contrast, the histidine-poor mutant exerted similar antimicrobial activities at both pH 6.2 and pH 7.6. At the physiological pH equivalent of fed female tick hemolymph (pH 6.2-6.4), histidine-rich samples had stronger antimicrobial activities than the histidine-poor mutant except against S. aureus. Generally, the native form was more active than either recombinant protein.
4. Differential expression of hebraein during blood-feeding of ticks
The results from protein profiling of hemolymph, analyzed by surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry combined with ProteinChip® technology (Fig. 1
) and RT-PCR (Fig. 2
) analysis, suggested that this antimicrobial protein was up-regulated by blood-feeding. This is also in accordance with our cloning strategy that used differential screening of our libraries with 32P[dCTP]-labeled cDNA probes.
|
|
CONCLUSIONS
Ticks have many opportunities to encounter microorganisms because of their special feeding behavior. Antimicrobial peptides/proteins are an innate immune system to counter pathogenic microorganisms. In terms of coevolution, perhaps ticks have developed multiple antimicrobial factors because they encounter a large diversity of pathogenic microbes including bacteria, fungi, and viruses from their hosts during their life cycle. With the growing problem of pathogenic organisms, which are resistant to conventional antibiotics, ticks are an emerging source of novel pharmaceutical substances to treat infections. Several defensin-like antimicrobial peptides, common in insects, have also been found in ticks. Hebraein is now the second type of antimicrobial protein isolated from ticks.
Hebraein has a unique primary structure that consists of six evenly distributed cysteine residues and a histidine cluster near its carboxyl terminus that contains eight histidine-aspartic or histidine-glycine continuous repeats. The lack of similarity to any known proteins in existing databases suggested that hebraein is a novel protein. Secondary structure prediction suggests that hebraein is a protein with multiple -helixes (4-6 -helixes) distributed evenly. This multi--helical structure is quite different from that of known antimicrobial proteins/peptides, many of which are peptides with a single -helix.
Hebraein’s novel primary structure, comprising six cysteine residues and a histidine-rich carboxyl-terminal region, suggested that it is a novel antimicrobial protein. However, when we were writing this paper, a cDNA sequence (GenBank accession number AY233212) cloned from another species of hard tick, Boophilus microplus, was published and the predicted protein was named microplusin. Microplusin has the same cysteine motif as hebraein, but has only four histidine residues near its carboxyl terminus. Aligning hebraein with microplusin gives an identity of 62% and a similarity of 73% between these two sequences, and our secondary structure prediction suggests that microplusin has four to six -helixes, as does hebraein. The high identity and similar secondary structure between these two proteins from two different hard ticks suggests they belong to the same protein family. Both members of this protein family have multiple histidine residues near their carboxyl terminus, suggesting that histidine residues play an important role; more proof is needed to support this hypothesis. Although known antimicrobial peptides/proteins have encompassed a wide variety of structural motifs, some new antimicrobial peptides/proteins with unique structural motifs are being found. The unique primary structure of hebraein and microplusin suggests that this family of proteins contains a novel structural motif.
Native hebraein and its recombinant forms displayed antimicrobial activities against the Gram-positive bacterium S. aureus, the Gram-negative bacterium E. coli, and the fungus C. glabrato, showing without doubt that hebraein is an antimicrobial protein with wide specificity. Compared with native and recombinant hebraein, the histidine-poor mutant protein displayed very weak antifungal activity in our experiments. This suggests that histidine residues in the primary structure play an important role in hebraein’s antifungal activity, although the carboxyl-terminal peptide fragment (NH2-HDHDHDHGHGHGHDHDPH-COOH) of hebraein did not display any detectable antimicrobial activity in our experiments. More work is needed to identify the actual role of the carboxyl-terminal histidine residues. It was noted that the antimicrobial activities of native and recombinant hebraein are substantially greater at pH 6.0 (physiological pH of engorged tick) than at pH 7.6, whereas the histidine-poor mutant exerted similar antimicrobial activities at both pH 6.0 and pH 7.6. It seems that hebraein exerts its activity in a pH-dependent manner, as has been found in other histidine-rich antimicrobial peptides such as histatin-like peptides. Histidine residues have a pKa of 
6.5, yielding histidine-rich peptides with high net positive charges at pH 6.0 yet relatively uncharged at pH 7.6. The positive charge of antimicrobial peptides undoubtedly facilitates their interactions with anionic microbial components such as lipopolysaccharide and increases their antimicrobial potency. When a tick blood feeds, its physiological environment becomes acidic; the acidic environment increases the positive charges on hebraein and possibly increases its antimicrobial potency. In terms of evolution, a histidine-rich antimicrobial protein like hebraein is very useful to selectively exert antimicrobial function during blood-feeding (Fig. 3
). The histidine-poor mutant still has high antimicrobial activity against S. aureus. These data may suggest that the activity against S. aureus is independent from the histidine cluster. Furthermore, hebraein showed antimicrobial activity against C. galbrato but not against C. albicans. This could mean that hebraein may work in different ways in different species.
|
Up-regulated expression of genes for other antimicrobial peptides (defensins), in response to blood-feeding has been observed in the blood-sucking insect, Stomoxys calcitrans and in the soft tick Ornithodoros moubata. The clone encoding hebraein was isolated from our forward differentially expressed cDNA library; its differential expression was further confirmed by RT-PCR and by SELDI-TOF mass spectrometry. When ticks attach onto their hosts, their body temperature and pH environment are changed; another obvious change is that ticks imbibe a large amount of blood from their hosts. One of these changes may provide the trigger leading to elevated hebraein expression. However, more work is necessary to identify the mechanism of regulation of hebraein expression.
Greater diversity of antimicrobial protein types makes understanding the mechanisms of antimicrobial action more complex, but at the same time provides more opportunities for designing novel and effective antimicrobial agents. 1) The discovery of tick hebraein with its all-helical structure composed of four to six -helixes adds a new member to the increasing family of antimicrobial proteins/peptides; 2) histidine-rich antimicrobial peptides with pH-dependent antimicrobial activity that can render them active at low pH and relatively inactive at neutral pH are advantageous during a tick’s blood-feeding when it encounters an acidic environment; 3) the pH-dependent properties of histidine-rich antimicrobial proteins may allow the design of agents that would function selectively in specific pH environments; for example, hebraein may be a useful template for developing antimicrobial peptides intended to function in acidic environments such as the gastric lumen or vagina of mammals; 4) the existence of hebraein in A. hebraeum and microplusin, its homologue in another ixodid tick, B. microplus, suggests there is another antimicrobial protein/peptide family that is specific for the tick’s innate immunity apart from the defensin-like peptides, although so far only two members of this new family have been found.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1154fje; doi: 10.1096/fj.03-1154fje
This article has been cited by other articles:
|
|
 |
|
  A. Makovitzki, A. Fink, and Y. Shai Suppression of Human Solid Tumor Growth in Mice by Intratumor and Systemic Inoculation of Histidine-Rich and pH-Dependent Host Defense-like Lytic Peptides Cancer Res., April 15, 2009; 69(8): 3458 - 3463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Gibson, F. R.T. Nelson, and D. Fyhrie On the Horizon From the ORS J. Am. Acad. Ortho. Surg., October 1, 2005; 13(6): 428 - 429. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
