Punkkien syljen metalloproteinaasit ja muut proteiinit

Muista silloij minua, kun punkki puree sinua.  (Vanha suomalainen sanonta) 

 Punkin puremaa EI tunne eikä  sitä huomaa, koska punkkisylki  on  niin puuduttavaa ainetta, mutta purematunne kestää siten myhemmässä vaiheessa  kauan  ilkeänä kuin "skorpionin pisto" eikä puremapaikan tuntoaistimus katoa viikkoihin,  ehkä pitempikin aika kuluu ja  puremakohta tuntuu kutiavalta  ja pistävältä, vaikka siitä olisi poistanut silmillä nähtävän  punkkimateriaalin.

  (Punkin sylki ja puremajärjestelmä ovat tämän takia kiinostavia.
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785505/)

TÄSSÄ artikkelissa: Eroista pehmeiden ja kovien punkkien syljen välillä
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211429/

Insect Biochem Mol Biol. 2008 Jan; 38(1): 42–58.

Published online 2007 Sep 25. doi:  10.1016/j.ibmb.2007.09.003

PMCID: PMC2211429 NIHMSID: NIHMS37009

Comparative sialomics between hard and soft ticks: Implications for the evolution of blood-feeding behavior

Ben J. Mans,^a John F. Andersen,^a Ivo M.B. Francischetti,^a Jesus G. Valenzuela,^a Tom G. Schwan,^b Van M. Pham,^a Mark K. Garfield,^c Carl H. Hammer,^c and José M.C. Ribeiro^a,*

Author information ► Copyright and License information ►

Abstract

Ticks evolved various mechanisms to modulate their host’s hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. 
 To address this issue the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks.
 The sialome is composed of ~130 secretory proteins, of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease  (MMPs)  families which also comprise the most abundant proteins found in the salivary glands.
 Comparative analysis indicates that the major protein families are conserved in hard and soft ticks.
 Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families.
In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.

Keywords: Argas, blood-feeding, evolution, proteome, sialome

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1. Introduction

Blood-feeding behavior evolved independently in insects in flies (Diptera), true bugs (Hemiptera), lice (Phthiraptera), fleas (Siphonaptera) and moths (Lepidoptera) (Ribeiro, 1995). In arachnids, hematophagous behavior evolved independently in ticks (Ixodida) and mesostigmatid mites (Mans and Neitz, 2004a; Radovsky, 1969). In each case the blood-feeding arthropod had to evolve mechanisms to modulate host defenses such as the hemostatic and immune systems. The mechanisms evolved by hematophagous arthropods mostly involve the secretion of salivary gland derived proteins during feeding and it is these proteins that target important host processes (Ribeiro, 1995). Adaptation to a blood-feeding environment by arthropods can thus be redefined as a study of the structural and functional evolution of the sialome (salivary gland proteomes of hematophagous organisms). From this perspective, the fact of independent adaptation to blood-feeding is clear when sialomes from different species are compared (Valenzuela et al., 2002a; Ribeiro and Francischetti, 2003; Champagne, 2004). In most cases the mechanisms to counteract the host’s immune and hemostatic defenses differ between various groups of hematophagous organisms.

 For the two major tick families, the soft (Argasidae) and hard (Ixodidae) ticks, this also holds and suggested that soft and hard ticks adapted to a blood-feeding environment independently (Mans et al., 2002a; Mans and Neitz, 2004a). Even so, phylogenetic analysis shows that the hard and soft tick families group as a monophyletic clade, indicating that they shared a common ancestor to the exclusion of other mites (Black and Piesman, 1994; Barker and Murrell, 2004). It was, however, also indicated that the ancestral tick lineage must have had some form of host-association, such as feeding on lymphatic fluid of the living host or as scavengers (Mans and Neitz, 2004a). This implies that the ancestral tick lineage must have features conserved in both families.

While the anti-hemostatic factors of soft and hard ticks differ in their mechanisms of action and protein families that they belong to, the two tick families share common protein folds in their salivary glands, such as the basic pancreatic trypsin inhibitor (BPTI) and lipocalin protein families (Mans and Neitz, 2004a).

 The BPTI/Kunitz family is composed of proteins that exhibit the basic pancreatic trypsin inhibitor fold (Laskowski and Kato, 1980).

 BPTI-like proteins act as thrombin, fXa and platelet aggregation inhibitors in soft ticks (Waxman et al., 1990; Karczewski et al., 1994; Van de Locht et al., 1998; Joubert et al., 1998; Mans et al., 2002b; Mans et al., 2007). 

In hard ticks, BPTI-like proteins inhibit the fVIIa/TF complex (Francischetti et al., 2002; Francischetti et al., 2004).

 The other major protein family so far described for ticks is the lipocalin family. In soft ticks, lipocalins function as anti-complement factors (Nunn et al., 2005), inhibitors of platelet aggregation (Waxman and Connolly 1993; Keller et al., 1993) and toxins (Mans et al., 2002c) and have been shown to be abundantly expressed in salivary glands (Mans et al., 2001; Mans et al., 2003; Oleaga et al., 2007). 

In hard ticks lipocalins that scavenge histamine and serotonin have also been described (Paesen et al., 1999; Paesen et al., 2000; Sangamnatdej et al., 2002).

 Recently high throughput sequencing of salivary gland transcripts from Ixodes scapularis and I. pacificus has shown that hard ticks possess more than 25 protein families in their sialomes (Valenzuela et al., 2002b; Francischetti et al., 2005; Ribeiro et al., 2006).

The question raised is how many of these protein families are also represented in soft ticks? 

Data on this could indicate the number of conserved protein families found in the salivary glands of the ancestral tick lineage and whether any specific orthologs existed between hard and soft ticks before their divergence.

To address this, the salivary gland transcriptome and proteome of the soft tick Argas monolakensis were characterized. A. monolakensis is limited to islands located on Mono Lake, California where it feeds annually on the breeding Californian gull population (Larus californicus) (Schwan et al., 1992). It is problematic for ornithologists working on the islands and is a potential vector of Mono Lake virus (Rheoviridae: Orbivirus) (Schwan and Winkler, 1984; Schwan et al., 1988). 

We show that the major protein families are conserved between hard and soft ticks. Even so, the numerous gene duplication events observed within individual protein families appear to be limited to lineage specific expansions, indicating that most of the sialome diversity observed for the different tick families evolved after their divergence.

3.9. The 8kDa cysteine-rich family is shared between hard and soft ticks

In some cases no positive hits were found between soft and hard ticks, for example, the 8kDa cysteine-rich family. However, small proteins might be divergent to an extent that even PSI-BLAST analysis might not detect homologies. In these cases, conserved features such as cysteine and disulphide bond patterns can be used to show that proteins possess the same fold. This study found at least 5 proteins that all share the same cysteine bond pattern but do not retrieve other proteins, i.e. the 8kDa cysteine-rich family. When compared to a group previously identified in Ixodes ticks (the ixodegrins), they clearly share the same cysteine pattern (Fig. 5). Some ixodegrins possess the RGD integrin recognition motif that correlates with that found for the snake derived disintegrin, dendroaspin and the hard tick platelet aggregation inhibitor variabilin (Francischetti et al., 2005). The Argas proteins, however, lack the RGD-motif. 

Proteins with the RGD-motif have been identified in the salivary glands of A. monolakensis and are orthologous to platelet aggregation inhibitors from the BPTI-like family found in the soft tick genus Ornithodoros (Mans et al., 2007).

• DENDROASPIN: https://www.ncbi.nlm.nih.gov/pubmed/7634091
• VARIABILIN:   http://www.jbc.org/content/271/30/17785

4. Discussion

The field of vector-host interaction has gained tremendously by high-throughput analysis of salivary gland transcripts and proteomes, collectively called the sialome (Ribeiro and Francischetti, 2003). This approach allows for the description of secretory products involved in blood-feeding of hematophagous organisms. Heretofore, the identification of proteins active at the blood-feeding site was only accessible using biochemical purification techniques to isolate bio-active components from salivary glands or saliva. The present advances in high-throughput methodologies allows us to gain a glimpse of the sialome in its full complexity. Thus, while biochemistry will always be needed to confirm and validate functional predictions derived from sialomic databases, the analysis of sialomes in terms of their protein domain compositions allows for a comparative analysis of sialome complexity and diversity that gives us insights into the evolution of blood-feeding behavior in arthropods. In the present study we analyzed the sialome of a soft tick species (A. monolakensis) and compare it with hard tick sialomes previously described. We derive the general conclusion that hard and soft ticks shared a similar salivary gland protein repertoire in their last common ancestor.

Approximately 130 potential secretory transcripts were identified in the salivary gland transcriptome of A. monolakensis by cDNA library construction. This compares well with the proteomic analysis using 2D-electrophoresis and liquid chromatography that resolved 78 and 118 abundant proteins, respectively. It also indicates that this number is probably close to the real number of secretory components found in the salivary glands of this soft tick.

 In comparison, more than 500 proteins have been described for the salivary glands of I. scapularis (Ribeiro et al., 2006). In this latter study, the data were obtained from the analysis of ~8000 ESTs, while the current study only analyzed ~3000 ESTs. However, these estimates likely correlate with salivary gland complexity, with hard tick salivary glands being more complex than that of soft ticks, as evidenced by that higher number of secretory acini and cell types found in hard ticks (Coons and Alberti, 1999). 

 Presumably, this is because hard ticks would need more components to control the host’s defense mechanisms and because they feed for longer periods of time and is exposed to the host’s immune system for extended periods of time.

The transcript numbers for highly abundant contigs also correlate well with the highly abundant proteins identified during the proteomic analysis. This correlation is consistent with the emerging paradigm in vector salivary gland biology, that proteins secreted during feeding are generally the most abundant salivary gland proteins with the correlated highest numbers of mRNA salivary gland transcripts. Even so, it is of interest that soft tick glands show such a good correlation between transcript and protein abundance. Soft tick salivary glands are normally filled with large secretory granules where proteins are stored until secretion during feeding (Roshdy, 1972, Roshdy and Coons, 1975, Coons and Roshdy, 1981, El Shoura, 1985; El Shoura 1987, Mans et al., 2004). Ticks, such as A. monolakensis, may only feed once a year and in the periods in between, granules will be stationary. Salivary glands can presumably only accommodate a certain number of granules and as such, proteins can be stored over prolonged periods of time and will accumulate, so that a general correlation between protein concentration and transcript number would not necessarily follow. Comparison of the cDNA libraries obtained for fed and unfed ticks do not differ markedly in terms of transcript numbers for various contigs (supplementary material). This would indicate that transcription occurs at the same rate for secretory transcripts regardless of the feeding status of the tick. Thus, if there is regulation of protein levels or salivary granule number, it would occur at a post-transcriptional level.

Analysis of the protein families identified in the Argas sialome indicate that they share to a large extent a similar salivary gland proteome with hard ticks. This would imply that most of the shared protein domains were also present in the ancestral tick lineage to the two families. Even so, few clear-cut orthologs were identified for highly abundant protein folds found in the soft and hard tick families. This observation could be extended to the lesser abundant protein families (results not shown). In the case of very short sequences (BPTI-kunitz and defensin families), sequences may evolve fast so that phylogenetic information is lost (Mans et al., 2002a). Host immune pressure and divergence times extending back to 400 MYA may also account for the high divergence of sequence (Barker and Murrell, 2004). Numerous biochemical studies have also shown that specific functions involved in the regulation of the host’s immune and hemostatic systems are not conserved between hard and soft ticks (Mans and Neitz, 2004a; Mans et al., 2007). The possibility thus exists that once functions are found for many of the divergent proteins in the sialomes; they will differ between the tick families. Those protein families for which orthologous proteins exist, are most probably ones conserved throughout invertebrates and would include the metalloproteases and anti-microbials. In short, these proteins would have had generalized functions before adaptation to a blood-feeding environment.

The predicted presence of certain shared protein folds in the ancestral tick lineage from which salivary gland function evolved raises the question as to where these folds derived from. For most of the major salivary gland protein families, they clearly derived from much more ancient members of the same folds that are present in arthropods. 

The ancestor to the salivary BPTI proteins probably derived from a hemolymph BPTI-like protein, such as those common in the hemolymph of arthropods (Mans and Neitz, 2004a). These proteins would generally be involved in the regulation of serine proteases involved in various processes and their presence in the salivary glands might have assisted in the inhibition of hemolymph proteases at a stage when the ancestral tick lineage still scavenged dead arthropods. 

Lipocalins most probably derived from a Lazarillo-like ancestor that was involved in the development of the neural system (Mans and Neitz, 2004b).  

• LIPOCALINS  https://en.wikipedia.org/wiki/Lipocalin

The metalloproteases are ancient enzymes that are conserved throughout the animal kingdom and are involved in all processes of extra-cellular matrix remodeling, a role it most probably also plays in arachnids. In the ancestral tick lineage these proteases must have played a role in digestion and liquefaction of a scavenged meal. It would seem a simple jump to re-adapt and retain them for blood-feeding use, especially when previous targets were collagen or fibrinogen-like. The presence of other “house-keeping, but adaptive” domains are also noted, for example the anti-microbials that would have a more ancient protective role, but would be co-opted for blood-feeding. Certain folds seem to be novel to tick salivary glands, most notably the BTSP-fold because of the high number of members found in both hard and soft tick salivary glands.

 The thrombospondin-repeat occurs in a number of mammalian proteins (Tucker, 2004). Thus far the only proteins outside of the BTSP family with TSP repeat that has been found in tick salivary glands are the ADAM-TS metalloproteases. This suggests that the BTSP fold and all its derivatives, i.e. 7DBF, 18kDa, 2CF, were most probably derived from a gene duplication of this domain from an existing metalloprotease. Whether this occurred more than once is difficult to ascertain, but would not be impossible, as the disulphide patterns from the 18.7kDa and 7DBF families are different and probably derived from existing TSP-1 families. Those domains which are currently orphan domains, i.e. proteins that cannot be assigned to any known protein family or domain, are most probably highly divergent members of known domains. It is foreseen that as more data become available, most of these orphan domains will eventually be assigned to well known families and that their origins will be easier to trace.

Certain recurring trends appear for many proteins found in tick salivary glands. They either belong to large families of which most genes seem to be lineage specific expansions, or their origins can be traced back to proteins that were already present in the salivary glands of the ancestral tick lineage. From this viewpoint, we propose that the ancestral tick lineage had a restricted set of salivary gland derived protein families which was not necessarily adapted to function within a blood-feeding environment. Subsequently the main tick families diverged and adapted to a blood-feeding environment. During this period, novel proteins involved in the modulation of the host’s hemostatic and immune systems evolved by gene duplication of full-length domains, as well as sub-domains. Proteins with functions that could affect the host’s defenses were also recruited during this period.