Phylogenetic relationships among several of these inhibitor families have been analysed: including the serpin family [2–8], Bowman–Birk [9,10], cereal trypsin⁄ a-amylase inhibitor [11],
Trang 1Evolutionary mechanisms acting on proteinase inhibitor variability
John T Christeller
Horticulture & Food Research Institute of NZ, Palmerston North, New Zealand
Proteinase inhibitors are a diverse group of proteins
that share not only a common biochemical activity,
but also the distinguishing feature of rapidly
undergo-ing evolutionary variation Currently, 59 distinct
famil-ies of proteinase inhibitors have been recognized [1] I
use the term ‘family’ in this review to denote these
phylogenetic groupings and the term ‘class’ to denote
inhibitors that interact with proteinases with
mechanis-tic similarities, i.e the serine-, cysteine-, asparmechanis-tic and
metallo-proteinases Phylogenetic relationships among
several of these inhibitor families have been analysed:
including the serpin family [2–8], Bowman–Birk [9,10],
cereal trypsin⁄ a-amylase inhibitor [11], proteinase
inhibitor I [12], proteinase inhibitor II [13] and cystatin
[14,15] Compared with the total number of families
that are currently recognized, this represents a very
small proportion, although phylogenetic trees for all
families have been constructed (http://merops.sanger ac.uk) These relationships are useful in developing an understanding of when and where the inhibitor class evolved; however, they do not provide information on the mechanisms driving gene evolution
The focus of many reviews of proteinase inhibitors over the last 25 years has been on classification and structure–function relationships These proteins have not been well recognized as a class of proteins with an interesting evolutionary history The purpose of this review is to summarize, for the first time, information relevant to proteinase inhibitor evolution, much of it collected incidentally, with the express intention of sti-mulating possible interest in this area
Proteinase inhibitors and their binding to proteinases have been extremely well characterized for more than
70 years and I focus only on those inhibitors that have
Keywords
evolution; hypervariability; pathogenesis;
predation; proteinase inhibitor
Correspondence
J T Christeller, HortResearch, Private Bag
11030, Palmerston North, New Zealand
Tel: +64 6356 8080, ext 7760
E-mail: jchristeller@hortresearch.co.nz
(Received 31 May 2005, accepted
15 September 2005)
doi:10.1111/j.1742-4658.2005.04975.x
The interaction of proteinase inhibitors produced, in most cases, by host organisms and the invasive proteinases of pathogens or parasites or the dietary proteinases of predators, results in an evolutionary ‘arms race’ of rapid and ongoing change in both interacting proteins The importance of these interactions in pathogenicity and predation is indicated by the high level and diversity of observable evolutionary activity that has been found
At the initial level of evolutionary change, recruitment of other functional protein-folding families has occurred, with the more recent evolution of one class of proteinase inhibitor from another, using the same mechanism and proteinase contact residues The combination of different inhibitor domains into a single molecule is also observed The basis from which vari-ation is possible is shown by the high rate of retention of gene duplicvari-ation events and by the associated process of inhibitory domain multiplication
At this level of reorganization, mutually exclusive splicing is also observed Finally, the major mechanism by which variation is achieved rapidly is hypervariation of contact residues, an almost ubiquitous feature of protein-ase inhibitors The diversity of evolutionary mechanisms in a single class of proteins is unlikely to be common, because few systems are under similar pressure to create variation Proteinase inhibitors are therefore a potential model system in which to study basic evolutionary process such as func-tional diversification
Trang 2the attributes of being involved in protein–protein
interactions representing antagonistic interorganism
interactions In this review, I draw attention to the
evi-dence that proteinase inhibitor evolution appears to
occur by multiple and interacting mechanisms not
cur-rently identified for other coevolving molecules and
that this feature may be indicative of both a high rate
of evolutionary change and the role of protein–protein
interactions Both attributes appear necessary, the
mechanisms are less apparent in proteinase inhibitors
targeted at intraorganism target proteinases than for
interorganism interactions, from which the majority of
the examples are drawn
Proteinase inhibitors, pathogens and
pests
Evolutionary pressures of various kinds have often
been hypothesized to cause active and rapid
evolution-ary change Various lines of evidence suggest that a
major function of proteinase inhibitors is to combat
the proteinases of pests and pathogens [16–20] The
secreted proteinases of the latter organisms are key
components of invasive cocktails, required for entry
into the host and rapid utilization of its constituent
proteins In these situations, there is clearly
evolution-ary pressure for the host to respond by evolving new
and effective inhibitors This model is often termed the
‘evolutionary arms race’ [21]
Consistent with the role of proteinase inhibitors in
resistance to invasion is the observation that massive
accumulation of proteinase inhibitors occurs in certain
tissues and organs that are likely sites of attack First
are those tissues whose high nutritional value presents
to a pest or pathogen the best possible site for attack,
for example, seeds [22], other plant storage organs
such as plant tubers [23,24], and the eggs of birds [25]
The reproductive strategies of these organisms require
that the best possible nutrition be provided in these
tissues and they are therefore clearly an attractive food
sources for others Proteinase inhibitors from these
sources have long been extensively studied and often
contain inhibitors of multiple families and classes
There appear to be few, if any, studies on the
protein-ase inhibitors in the eggs of other egg-laying organisms
such as fish and insects, or on the proteinase inhibitors
of organisms that retain their eggs internally It is
possible that the evolution of internal egg-bearing is
related to the reduced pest, pathogen and parasite
attack that comes with this strategy
The second attractive site of attack by invasive
organisms is fluids that permit transport of the
patho-gen throughout the host, for example, mammalian
serum, invertebrate haemolymph and plant phloem Indeed, these three fluids are, once again, a rich source
of many and varied proteinase inhibitors and have been studied extensively However, caution needs to be applied; the primary role of these inhibitors in blood appears to be regulation of the blood-clotting cascade
In insects, proteinase inhibitors in the haemolymph clearly play a part in the immune response, partic-ularly in regulating the activation of prophenoloxidase
in response to invasion by pathogens [26] The func-tion of the diverse phloem proteinase inhibitors is unclear [27–30]
The third situation that can be identified in which extensive and varied proteinase inhibitor accumulation arises is the reverse situation, where proteinase inhibi-tors themselves are the pathogenic determinants For example, the salivas of leeches and blood-sucking insects contain multiple inhibitors [31] that inactivate the proteinases of the blood-clotting cascade, thereby preventing blood-clotting and permitting the invader
to feed freely
Structural gene evolution
In this review, the evolutionary mechanisms used by organisms to enhance variation in the structural genes
of proteinase inhibitors are discussed This story is but one third of the equation; the other two parts, evolu-tion of cognate proteinases and evoluevolu-tion of the pro-teinase inhibitor regulatory sequences are not discussed except en passant, partly because of the need to limit the breadth of the review and partly because the iden-tification of the cognate proteinase is, in many cases, yet to be verified and because mechanisms of promoter evolution are a distinct and new topic entirely The identification of cognate proteinases is often extremely challenging and, where achieved, has led to specific studies on coevolution [19,32,33] The evolution of promoters has clearly occurred with equal rapidity to that of the structural genes For example, orthologues
of the proteinase inhibitor I family are known that have constitutive, tuber-specific [34], wounding signal-induced, leaf-specific [35], phloem-specific [36], fruit-specific [37], developmentally regulated [38], ethy-lene-induced [39] and cell-cycle-specific [40] promoters These processes may be most active in plants because many pests and pathogens use S1 superfamily serine proteinases as pathogenic determinants and these are not common in plants (The Arabidopsis Information Resource, TAIR, database at http://www arabidopsis.org, lists 55 serine proteinases from 546 endopeptidases); thus the problem of isolating serine proteinase inhibitors from plant metabolic and
Trang 3regulatory processes is less critical, a consideration that
might slow inhibitor evolution in other organisms In
mammals, serpins separate into secreted inhibitors
involved in pathogen defence processes and
intracellu-lar inhibitors involved in celluintracellu-lar regulation; the former
show high rates of evolution, whereas the latter do
not [8]
In the following sections, each distinct evolutionary
mechanism is discussed separately, yet it is clear from
the examples given that they do not act in isolation
The first three mechanisms relate to the fundamental
evolution of this group of molecules The fourth to the
sixth mechanisms appear to establish a higher level of
diversity that forms the basis from which the seventh
and final mechanism described is fundamental for
generation of the large variation observed
Recruitment of other protein-folding
scaffolds to proteinase inhibitor
function
It is apparent that recruitment of numerous
protein-folding scaffolds to proteinase inhibitor function has
occurred This is clearly seen in the various folds of
several proteinase inhibitors
First, inhibitory serpins share 30% amino acid
sequence homology with ovalbumin, the major storage
protein of egg white, and share, with several other
noninhibitory proteins, the same basic structure [41]
Second, five members of the large cereal a-amylase
inhibitor family have developed proteinase inhibitor
function, and three of these have lost the former
activ-ity during evolution [11] Interestingly, these inhibitors
exhibit the same novel backbone structure as is also
observed in 2S seed storage proteins and in nonspecific
lipid transfer proteins [11,42], suggesting even earlier
recruitment of a-amylase inhibitor function Third,
equistatin [43,44], fish egg inhibitor [45], saxiphilin
[46], testican [47] and p41 major histocompatibility
complex fragment [48] are cysteine proteinase
inhibi-tors based on the thyroglobulin fold Fourth, soybean
Kunitz inhibitors have homology to noninhibitory
pro-teins sporamin A [49], stress-induced propro-teins [50],
dehydroascorbate reductase [51] and miraculin [52]
Finally, it has been suggested that both Bowman–Birk
and cystatin inhibitors have evolved from an ancient
ribonuclease-like gene [53]
Other major plant storage proteins, as well as the
cereal a-amylase inhibitor and the soybean Kunitz
inhibitor may have been recruited to additional
func-tion For example, the Bowman–Birk inhibitors in
seeds and proteinase inhibitors I and II in potato
tubers are present at such high levels that they
function as storage proteins [23] Possibly their ances-tral types, lacking proteinase inhibitor function, have been lost due to the evolutionary advantage of produ-cing dual-function proteins Thus, although we have only identified five examples from 59 known inhibitor classes [1] it is possible that further examples will be identified in the future as additional proteinase inhib-itor structures become available
Knowledge of the inhibitory mechanism may assist understanding of how the recruitment processes evolved Two of the above example inhibitors are serine proteinase inhibitors that use the ‘standard canonical’
or ‘Laskowski mechanism’ This involves the presence
on the surface of the inhibitor of a stabilized loop that can mimic a substrate but which has long residency times in the proteinase active site pocket as a result of that conformational stability The loop also has a pro-truding amino acid side chain that mimics the protein-ase target specificity [54–56] The combination of these features produces a rapid-binding, slow-release specific inhibitor rather than a substrate The distinction may,
in fact, not be clear cut, as substrate proteins may, under specific conditions, have inhibitory properties rel-ative to small reporter substrates used to assay protein-ases, for example, napins, legumins [57] (W A Laing, HortResearch, personal communication)
It seems entirely possible that any surface loop or even stretch of exposed amino acids could evolve inhibitory function by reducing flexibility through evol-ving intraprotein interactions, creating a loop by inser-tion of a very few amino acids and mutating a single amino acid to form a P1 bait site residue Evidence for this can be found by inspection of crystal structures of inhibitors and their putative ancestral proteins This is clear for the cereal trypsin⁄ amylase family in which the structure of the bifunctional ragi inhibitor complex with Tenebrio molitor a-amylase [58] showed that the proteinase binding loop adopts a canonical conforma-tion at the opposite side of the molecule This loop is absent from the a-amylase inhibitor [59] It also appears correct for serpins, because the intact active site loop adopts a distorted helical conformation in the P10–P3¢ region, overlapping a type I b-turn in P2¢–P5¢ [60] compared with an undistorted a-helix for ovalbu-min [41] The modification in structure between inhibi-tory and noninhibiinhibi-tory forms is probably due to the presence of proline residues and a four amino acid insertion at this point in the inhibitor sequence [60] This hypothesis requires that the rapid evolution of proteinase inhibitors began somewhat late in evo-lutionary history, when many of the major protein folds had already evolved, and that evolution has been from established essential functional proteins to new
Trang 4supplementary functions If the stimulus for this to
occur is the development of pests and pathogens it
supposes that these interactions also were not a feature
of early life and, so far as the discussion revolves
around multicellular organisms as hosts, this is a
rea-sonable assumption
It should be noted, however, that the ‘Laskowski
mechanism’ is not a feature of most families of
inhibi-tors that inhibit proteinase classes, i.e inhibiinhibi-tors of
metalloproteinases, cysteine proteinases and aspartic
proteinases even though these proteinases do possess
characteristic residues at their active sites (cation,
sul-fydryl and aspartyl, respectively) Although many of
these inhibitors are competitive, binding at the active
site to prevent access to substrates, they do not mimic
a substrate, are not cleaved reversibly and often utilize
more than a single exposed loop in their direct active
site contacts Of these proteins, only one class, the
thy-ropins, has been identified as recruited structures
Change of inhibitory class
Recruitment of one family of proteinase inhibitors to
inhibit a second class of proteinases is an
evolution-ary mechanism that can be readily identified and for
which several examples are known It is a special case
of recruitment of another protein fold where the
pro-teinase inhibitory structure is also recruited, or is
likely to have been recruited, and is likely to have
occurred more recently This is seen in (a) serine
pro-teinase inhibitors of the serpin family recruited to
cys-teine proteinase inhibition [61], (b) serine proteinase
inhibitor of the serpin family recruited to aspartic
proteinase inhibition [3,62], (c) serine proteinase
inhibitor of the seed Kunitz family recruited to
cys-teine proteinase inhibition [63], (d) serine proteinase
inhibitor of the seed Kunitz family recruited to
aspar-tic proteinase inhibition [64], (e) serine proteinase
inhibitor of the Bowman–Birk class recruited to
cys-teine proteinase inhibition [65], (f) cyscys-teine proteinase
inhibitors of the cystatin family recruited to aspartic
proteinase inhibition (W A Laing, unpublished
observations), and (g) cysteine proteinase inhibitors of
the thyropin class, recruited to aspartic proteinase
inhibition [66] This recruitment mechanism is not
uncommon, having been identified in six of 59 known
inhibitor families to date
It is necessary to discuss each case individually,
rather than assume that this represents a special and
recent example of recruitment of another functional
protein as discussed above The serine proteinase
inhibitors of the serpin family do not operate via the
‘Laskowski mechanism’ However, they have evolved a
very effective ‘suicide’ irreversible mechanism in which the inhibitor (following Michelis complex formation) partitions between a tetrahedral stable intermediate and a cleaved, inactive inhibitor [67,68] leading to covalent bond formation via acyl bond formation and
a large conformation This is often observed as an SDS-stable product that migrates more slowly during PAGE than the unreacted proteinase and inhibitor It seems that this mechanism has been maintained during evolution to the cysteine inhibitory form because the structure with a reactive site loop remains [69,70], the corresponding thioacyl complex has been detected [71] and the cleavage site remains within the single reactive loop of several dual class inhibitors [72,73]
Because the serine proteinase inhibitor molecule is apparently found in a diverse range of organisms: euk-arya, bacteria, archaea and viruses [6] it is clearly the ancestral form and appears to have evolved directly to the new class of cysteine proteinase inhibitors found,
to date, in mammals and viruses [61] The situation with the aspartic proteinase inhibitors has not yet been resolved with no studies investigating the presence of a covalent bond being reported Interestingly, both these examples of recruitment of function appear to be an old divergence, occurring at a similar time to mamma-lian divergence [73] although these altered inhibitors have, to date, been reported only from mammals It is also noteworthy that whereas most serpins are secre-ted, one clade is intracellular and members function as regulators of ‘promiscuous’ proteinases [8], rather than being involved regulation of endogenous cascades or in protection from pathogens
Despite extensive studies, we still do not know whe-ther the aspartic proteinase inhibitors that have clearly been recruited from standard mechanism Kunitz seed serine inhibitors, have recruited the latter molecules’ serine proteinase inhibitor loop to inhibit aspartic pro-teinases At least one member of this small family restricted to Solanaceae [64] has the ability to inhibit both classes [74] Clearly, a structure of an inhibitor– aspartic protease complex would be of great interest because successful attempts to determine a cleavage site for this system have not been reported Owing to the very different pH optima for complex formation of the two classes it is difficult to even show whether sim-ultaneous binding of two proteinases is possible Although the active site loop of serine proteinase inhibitors of the seed Kunitz appears to have been dis-rupted in the cysteine inhibitors of this class [63], there
is no information on the mode of interaction of these inhibitors with cysteine proteinases although some members of these inhibitors retain weak antitrypsin and antichymotrypsin activity [75]
Trang 5The cysteine proteinase inhibitor, bromelain
inhib-itor VI from pineapple, is a double-chain inhibinhib-itor that
shares similar folding and disulfide bond connectivities
with the Bowman–Birk trypsin⁄ chymotrypsin inhibitor
[65] The authors suggested that these inhibitors have
evolved from a common ancestor and differentiated in
function during a course of molecular evolution
How-ever, the B-domain of bromelain inhibitor VI has weak
antitryptic activity, suggesting that class conversion is
a reasonable alternative
The recruitment of a cystatin to aspartic proteinase
inhibitor [28,76] is based on sequence homology of
around 30% and similarity of around 50% between
rice cystatin and squash aspartic proteinase inhibitor
(SQAPI) and modelling the later onto the crystal
struc-ture of the former (W A Laing, unpublished
observa-tions) Inhibition appears to involve the same areas of
interaction; mutation and removal of residues at two
regions known to be involved in cystatin interactions
[77] abolishing aspartic protein inhibition (P Farley,
unpublished data) and hypervariability within the small
inhibitor family also occurs at these sites and at the third
site known to be involved in cystatin interactions (J T
Christeller, unpublished observations) The tentative
conclusion is that this small family of aspartic
protein-ase inhibitors, restricted to members of the Cucurbitales
(J T Christeller, unpublished data) has evolved directly
from the much more widespread cystatin inhibitors
Equistatin is a protein consisting of three
thyroglob-ulin domains [44], the N-terminal domain inhibiting
cysteine proteinases and the central and C-terminal
domains inhibiting aspartic proteinases Equistatin is
therefore a member of the thyropin class of inhibitors,
of which all other known members are cysteine
proteinase inhibitors The published structure of the
thyropin p41 fragment shows a wedge shape and
three-loop inhibitory structure similar to cystatins, thus
suggestive of convergent evolution [78] There is no
information on the mode of inhibition of the aspartic
proteinase inhibitor variant domains of equistatin
Proteinaceous aspartic proteinase inhibitors are very
rare in nature and four of the six known families are
recruited from other classes, with only two, the yeast
inhibitor IA3 [79] and the Ascaris PI-3 inhibitor [80]
being uniquely aspartic inhibitor families Both
inhibi-tors are small and have quite idiosyncratic inhibitory
mechanisms Thus inhibitor class change appears to be
the mechanism of choice for this class of proteinases
These observations, combined with their rarity, may
indicate that the aspartic class of proteinases have
evolved relatively recently, at least to fulfil the function
of defence proteins Given the relative rarity of
metal-loproteinase inhibitors, similar questions may be asked
about the time of their evolution and their recruitment
to a defence protein role
Domain shuffling Recently, two examples of multidomain proteins have been identified in which different domains with distinct inhibitor classes are fused into a single inhibitor First, testican is a multidomain protein with three domains having homology to different proteinase inhibitors, an N-terminal domain with metalloproteinase inhibitory activity [81], a follistatin-like domain with similarity to Kazal serine proteinase inhibitors although no serine proteinase inhibition has yet been reported and a thy-ropin domain that inhibits cysteine proteinases [47] Second, the WFIKKN protein [82] has, based on homology, a whey acidic protein metalloproteinase inhibitor module, a follistatin⁄ Kazal inhibitor module and two Kunitz-type modules One of the latter domains has been shown to inhibit trypsin [83] Although this evolutionary mechanism seems to be uncommon, the existence of this type of domain-shuf-fling event may indicate the high evolutionary pressure that proteinase inhibitors experience, permitting coordinate expression of proteinase inhibitors against a cocktail of secreted proteinases
Gene duplication Gene duplication is a very common feature of protein-ase inhibitors Many, if not most, inhibitors are pre-sent as small gene families with altered specificities among the paralogues Two features of pathogenicity and predation may drive this process; first, the pres-ence of multiple attacking organisms with variation in their proteinases and, second, the presence of multiple proteinases in single organisms, for example, secreted aspartic proteinases of Candida number more than 10, whereas the digestive proteinase genes of lepidopteran larvae number in the hundreds Thus gene duplication, coupled with rapid adaptation processes (detailed below) form a mechanism to resist invaders Because there is no evidence that these genes duplicate at higher rates than any others, their fixation in the gen-ome is more likely to be due to rapid adaptation and the selective advantage obtained Fryxell [84] considers that fixation of gene duplication is maintained by co-evolution of functionally related gene families His hypothesis seems appropriate for proteinases and their inhibitors Habu et al [85] studied the evolution of duplicated Kunitz inhibitor genes in winged bean and
a complex series of gene inactivation and gene conver-sion events was inferred
Trang 6Domain replication and circular
permutation
Domain replication serves a similar function to gene
duplication, both providing a base from which
vari-ability can be established Instead of complete gene
duplication, including promoter and terminator
sequences and possible reintegration at a distinct
locus, there is duplication of the inhibitory domain
sequence with the domains remaining fused This
evo-lutionary mechanism is very common in many
inhib-itor gene families and has been well reviewed for
some time [1,55,86] Again, it serves the function of
not only coordinated expression, but also increased
levels of expression The most extreme examples are
genes in the proteinase inhibitor II and cystatin
famil-ies Proteinase inhibitor II family members have been
characterized with varying numbers of domains, from
one [13,87], two [88], three [89], four [90], six [91]
through to eight [92] Many of these proteinase
inhib-itor II polyproteins are processed proteolytically,
dis-playing the highly unusual phenomenon of cleavage
within the domain [93], with the final molecule having
N- and C-terminal sections being circularly
permuta-ted The final conformation adopted by these domain
hybrid molecules is identical to that adopted by single
domain versions [87], likely to represent the putative
ancestral sequence order [94] A mechanism involving
unequal gene cross-over has been proposed to
account for this variation by Barta et al [13] who
also noted that this may be a scenario to enhance
functional diversity against pathogens These
multi-plication and circular-permutation events have been
followed by rapid divergence within single genes to
target diverse proteinases Members of the proteinase
inhibitor II family have been reported to inhibit
chymotrypsin, elastase, oryzin, pronase E, subtilisin
and trypsin [95–97] Circular permutation has been
observed in other proteins, including proteinases
[98,99]
The second extreme example occurs in potato tubers
where a single 85 kDa polypeptide, potato
multicysta-tin comprises eight tandem cystamulticysta-tin domains, with
53–89% identity of residues, linked by proteolytically
sensitive junctions [100,101] Potato multicystatin
com-prises a family of four to six genes in potato and the
pattern of gene expression, as well as the properties of
the protein suggest that potato multicystatin has a role
in plant defence [101] Although single domain
cysta-tins are most common, a three-domain multicystatin
has been isolated from sunflower seeds [102] and
kinin-ogens are also three-domain cystatins [103]
Mutually exclusive splicing
A single serpin gene of Manduca sexta expressed in the haemolymph comprises 10 exons, with the ninth, containing the reactive site loop, existing as 12 vari-ants all positioned between the eighth and tenth exons [103–105] All 12 variants, each possessing a single ninth exon, are found in a M sexta cDNA lib-rary, indicating that mutually exclusive exon splicing
is occurring The mechanism occurs in many other other genes, being first reported in tropomyosin [106] and includes serpin genes from Bombyx mori [107], Ctenocephalides felis [108], Drosophila melanogaster and Caenorhabditis elegans [109]; albeit with smaller numbers of exclusive exons The only other proteinase inhibitors reported to use alternative splicing are mammalian calpastatins [110] in which the variant exons include the initiation codons It is likely that the evolution of the system occurs by uneven cross-over of chromosomes [105]
Hypervariability Hypervariability in proteins, which may be defined as enhanced variation among orthologous and paralogous genes at the contact residues, is often proposed as an example of positive Darwinian evolution although this idea remains controversial [111] Hypervariability is virtually a defining feature among proteinase inhibitors and the key mechanism in creating variation Hyper-variability in proteinase inhibitors occurs when nucleo-tides encoding proteinase contact residues within the active site loops in ‘Laskowski mechanism’ inhibitors such as the ovomucoids [112,113] and the aprotinin family [32] and in ‘non-Laskowski (nonstandard)’ inhibitors such as the serpins [17,114,115] mutate and are fixed in the genome at a much higher rate as amino acid variants compared with residues elsewhere
in the molecule [32], or, at a nucleotide level, at a rate higher than silent mutations [116] That hypervariation results in functional diversity, although long the pre-dicted outcome, has been demonstrated by Barbour
et al [117] The importance of the three preceding mechanisms that act to produce the gene multiplicity needed to generate functional diversity cannot be over-emphasized Ohta [33] also concluded that ‘that posit-ive selection operated after duplication to increase functional diversity’ and concluded that
mechanistical-ly ‘hypervariability of amino acids at the reactive cen-ter is generated by an incen-teraction among natural selection, random genetic drift, point mutation, and gene conversion’
Trang 7Proteinase inhibitors from classes other than serine
proteinase inhibitors have also shown hypervariability,
such as cystatins [32] and squash aspartic proteinase
inhibitor (SQAPI; J T Christeller, unpublished data)
Hypervariability is extremely common among
protein-ase inhibitors and proteinprotein-ases [32, 33,118], with only
two examples of diversification of proteinase inhibitors
without evidence of positive selection have been
repor-ted [12,119] Hypervariability appears to be a feature
of other pairs of interacting molecules such as
resist-ance genes [120], surface antigens [121], thionins [122]
and conotoxins [111,123], but is otherwise rare, in line
with the neutral model of evolution [124] These
inter-actions also seem to be examples of the operation of a
coevolutionary ‘arms race’ Whether these proteins will
also show a similar range of evolutionary mechanisms
as proteinase inhibitors in addition to hypervariability
is not yet known, although as avirulence genes are
being identified, some information on their evolution is
being published [125–128]
Although the above description completes the
speci-fic evolutionary mechanisms currently known to occur
in proteinase inhibitors, there are two additional
rela-ted areas that are relevant to proteinase inhibitor
evolution
Interaction of proteinase inhibitors
with inactive proteinases
An emerging story is the possibility of proteinase
inhibitor inactivation by inactive variants of
pro-teinases as antagonists Mathialagan and Hansen [62]
suggest that the pregnancy-associated glycoproteins
that are inactive aspartic proteinases [118] may be the
cognate proteinases of uterine serpins The inactive
proteinases would act to bind a proportion of the
inhibitors, leaving some active proteinases to fulfil the
desired function, in a situation where overexpression
of active proteinases is itself undesirable A similar role
has been suggested for the multigene families of
inacti-vated serine and cysteine proteases in Sarcoptes scabiei
[129,130], i.e antagonists of host proteinase inhibitors
Both stories may represent an adaptation to the
para-sitic interactions involved in pregnancy and scabies
infection This system may operate elsewhere For
example, there are inactive proteinase cDNAs in insect
midgut, both induced [131] and constitutively
expressed [132] If these transcripts are translated and
are active in binding proteinase inhibitors, then they
may have a role in insect resistance [132] and in
explaining the patterns of adaptation observed when
insects are fed diets containing proteinase inhibitors
[133–135] This inundative strategy, a biochemical
‘male sterile technique’, may be relatively uncommon because it requires additional resources in terms of protein synthesis However, it also presents a new chal-lenge for proteinase inhibitor evolution because the mechanisms described above operate on the evolving proteinase specificity and structural changes rather than the cryptic changes occurring in proteinase inacti-vation
Proteinase inhibitors and parasitism Our discussion so far has described the various evolu-tionary mechanisms that have been observed in prote-inase inhibitors in their coevolutionary variation with cognate proteinases The examples used exclusively illustrate the concept of a causal relationship between this process and successful plant predation and patho-genesis It is reasonable to interpret parasitism as a less extreme form of pathogenesis or predation in which the host is maintained in a live state The literature on the determinants for successfully establishing a parasi-tic relationship includes many examples in which pro-teinases have been implicated These include: (a) stress-induced ClpP of Listeria monocytogenes and its crucial role in intracellular survival of this pathogen [136], (b) inactivation of serine proteases in the Scabies mite [129], (c) mycoparasitism of Agaricus bisporus [137], (d) phytoplasma virulence [138], (e) serine proteinase inhibitors may play a role in the tick larvae fixation and feeding processes [139], (f) Trypanosoma cruzi infection [140], and (g) hookworm adaptation [141] It
is therefore possible that in these situations the same causal relationship exists between proteinase–protein-ase inhibitor evolution and parasitism However, the existence of proteinase inhibitors, let alone variation and adaptation, has not been demonstrated in all these parasitic relationships We can speculate that this rela-tionship has more far-reaching implications Parasitism has been implicated as a driving force in the develop-ment of sex [141–143] If this is true then perhaps pro-teinase inhibitor evolution is even more important they previously recognized
Conclusion The evolutionary pressure surrounding the interaction
of proteinases and their inhibitors in an antagonist environment seems to be immense The impression left
by the survey presented here is that inhibitors are using virtually every trick in the evolutionary book, and sometimes in combination, to create variation and that the various mechanisms occurring do so in a ran-dom fashion Whether there are new inhibitors and
Trang 8adaptive mechanisms yet to be discovered does not
diminish this already impressive list It is probable that
proteinase inhibitors are one of the most actively
evol-ving proteins and that they deserve further
considera-tion as model systems to study important evoluconsidera-tionary
phenomena
Acknowledgements
I would like to thank W A Laing (HortResearch,
Auckland, New Zealand) for reading the manuscript
The project was supported by funding from the Public
Good Science Fund, administered by the New Zealand
Foundation for Research, Science and Technology
(Contract C06X0207)
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